Cloning and expression of Haemophilus somnus transferrin-binding proteins

ABSTRACT

Cloning and expression of genes encoding  H. somnus  transferrin-binding proteins are described. The transferrin-binding proteins can be used in vaccine compositions for the prevention and treatment of  H. somnus  infections, as well as in diagnostic methods for determining the presence of  H. somnus  infections.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/267,749, filed Mar. 10, 1999 from which priorityis claimed under 35 USC §120 and which application is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to bacterial antigens andgenes encoding the same. More particularly, the present inventionpertains to the cloning, expression and characterization oftransferrin-binding proteins from Haemophilus somnus (H. somnus) and theuse of the same in vaccine compositions. BACKGROUND

[0003]Haemophilus somnus is a Gram-negative bacterium which causes anumber of disease syndromes in cattle, collectively referred to asbovine hemophilosis. The bacterium is commonly associated withthromboembolic meningoencephalitis (ITEME), myocarditis, septicemia,arthritis, and pneumonia (Corbeil, L. B. (1990) Can. J. Vet. Res.54:S57-S62; Harris and Janzen (1990) Can. Vet. J. 30:816-822; Humphreyand Stephens (1983) Vet. Bull. 53:987-1004). These diseases causesignificant economic losses to the farm industry annually.

[0004] Conventional vaccines against H. somnus infection are eitherbased on killed whole cells or on a protein fraction enriched in outermembrane proteins (OMPs). However, whole cell bacterins and surfaceprotein extracts often contain immunosuppressive components which canrender animals more susceptible to infection. Recombinant vaccinescontaining H. somnus lipoproteins, LppA, LppB and LppC, have beendescribed. See, e.g., International Publication No. WO 93/21323,published Oct. 28, 1993. However, there remains a need for efficacioussubunit vaccines against H. somnus infection.

[0005] Iron is an essential element for growth of most microbes.Weinberg, E.D. (1978) Microbiol. Rev. 42:45-66. Even though iron isabundant within mammalian tissues, virtually all iron within themammalian body is held intracellularly as ferritin or as heme compounds,pools which are generally inaccessible to invading microorganisms.Additionally, the small amount of iron present in extracellular spacesis effectively chelated by high-affinity iron-binding host glycoproteinssuch as transferrin, present in serum and lymph, and lactoferrin,present in secretory fluids and milk. Otto et al. (1992) Crit. Rev.Microbiol. 18:217-233.

[0006] Hence, bacterial pathogens have developed specific iron-uptakemechanisms. In many bacterial species, these mechanisms involve thesynthesis and secretion of small compounds called siderophores whichdisplay high affinity for ferric iron (FeIII). Siderophores are capableof removing transferrin-bound iron to form ferrisiderophore complexeswhich in turn are recognized by specific iron-repressible membranereceptors and internalized into the bacterium where the iron isreleased. Crosa, J. H. (1989) Microbiol. Rev. 53:517-530. Somegram-negative bacteria do not secrete detectable siderophores when grownin an iron-deficient environment but produce outer membrane proteinsthat bind directly and specifically to transferrin, thereby allowingiron transport into the bacterial cell. Transferrin binding proteinstend to be highly specific for the transferrin of their natural host.The ability of microorganisms to bind and utilize transferrin as a soleiron source, as well as the correlation between virulence and theability to scavenge iron from the host, has been shown (Archibald andDeVoe (1979) FEMS Microbiol. Lett. 6:159-162; Archibald and DeVoe (1980)Infect. Immun. 27:322-334; Herrington and Sparling (1985) Infect. Immun.48:248-251; Weinberg, E. D. (1978) Microbiol. Rev. 42:45-66).

[0007] Two transferrin-binding proteins, termed transferrin-bindingprotein 1 and 2 (Tbp1 and Tbp2), respectively, have been identified inbacterial outer membranes. For example, Gonzalez et al. (1990) Mol.Microbiol. 4:1173-1179, describes 105 and 56 kDa proteins fromActinobacillus pleuropneumoniae, designated porcine transferrin bindingprotein 1 (pTfBP1) and porcine transferrin binding protein 2 (pTfBP2),respectively. U.S. Pat. Nos. 5,417,971, 5,521,072 and 5,801,018 describethe cloning and expression of two transferrin binding proteins from A.pleuropneumoniae, as well as the use of the proteins in vaccinecompositions. Schryvers, A. B. (1989) J. Med. Microbiol. 29:121-130,describes two putative transferrin-binding proteins isolated fromHaemophilus influenzae. U.S. Pat. No. 5,708,149 and InternationalPublication No. WO 95/13370, published May 18, 1995, describe therecombinant production of H. influenzae Tbp1 and Tbp2. U.S. Pat. Nos.5,141,743 and 5,292,869 and International Publication No. WO 90/12591describe the isolation of transferrin-receptor proteins from Neisseriameningitidis and the use of the isolated proteins in vaccinecompositions. International Publication No. WO 95/33049, published Dec.7, 1995, and European Publication No. EP 586,266, describe DNA encodingN. meningitidis transferrin binding proteins. Finally, Ogunnariwo et al.(1990) Microbiol. Path. 9:397-406, describe the isolation of twotransferrin-binding proteins from H. somnus.

[0008] However, to date, the transferrin binding proteins from H. somnushave not been recombinantly produced.

DISCLOSURE OF THE INVENTION

[0009] The present invention is based on the discovery of genes encodingH. somnus transferrin-binding proteins and the characterization thereof.The proteins encoded by the genes have been recombinantly produced andthese proteins, immunogenic fragments and analogs thereof, and/orchimeric proteins including the same, can be used, either alone or incombination with other H. somnus antigens, in novel subunit vaccines toprovide protection from bacterial infection in mammalian subjects.

[0010] Accordingly, in one embodiment, the subject invention is directedto an isolated nucleic acid molecule comprising a coding sequence for animmunogenic H. somnus transferrin-binding protein selected from thegroup consisting of (a) an H. somnus transferrin-binding protein 1 and(b) an H. somnus transferrin-binding protein 2, or a fragment of thenucleic acid molecule comprising at least 15 nucleotides.

[0011] In additional embodiments, the invention is directed torecombinant vectors including the nucleic acid molecules, host cellstransformed with these vectors, and methods of recombinantly producingH. somnus transferrin-binding proteins.

[0012] In still further embodiments, the subject invention is directedto vaccine compositions comprising a pharmaceutically acceptable vehicleand an immunogenic H. somnus transferrin-binding protein selected fromthe group consisting of (a) an H. somnus transferrin-binding protein 1,(b) an H. somnus transferrin-binding protein 2 and (c) an immunogenicfragment of (a) or (b) comprising at least 5 amino acids, as well asmethods of preparing the vaccine compositions.

[0013] In yet other embodiments, the present invention is directed tomethods of treating or preventing H. somnus infections in a mammaliansubject. The method comprises administering to the subject atherapeutically effective amount of the above vaccine compositions.

[0014] In additional embodiments, the invention is directed to methodsof detecting H. somnus antibodies in a biological sample comprising:

[0015] (a) providing a biological sample;

[0016] (b) reacting the biological sample with an immunogenic H. somnustransferrin binding protein selected from the group consisting of (a) anH. somnus transferrin-binding protein 1, (b) an H. somnustransferrin-binding protein 2 and (c) an immunogenic fragment of (a) or(b) comprising at least 5 amino acids, under conditions which allow H.somnus antibodies, when present in the biological sample, to bind to theH. somnus transferrin-binding protein to form an antibody/antigencomplex; and

[0017] (c) detecting the presence or absence of the complex,

[0018] thereby detecting the presence or absence of H. somnus antibodiesin the sample.

[0019] In yet further embodiments, the invention is directed to animmunodiagnostic test kit for detecting H. somnus infection. The testkit comprises an H. somnus transferrin-binding protein selected from thegroup consisting of (a) an H. somnus transferrin-binding protein 1, (b)an H. somnus transferrin-binding protein 2 and (c) an immunogenicfragment of (a) or (b) comprising at least 5 amino acids, andinstructions for conducting the immunodiagnostic test.

[0020] These and other embodiments of the present invention will readilyoccur to those of ordinary skill in the art in view of the disclosureherein.

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIGS. 1A-1B show the nucleotide sequences of the H. somnus tbp1and tbp2 genes (SEQ ID NO:1). The tbp1 gene is found at positions2891-5803 and the tbp2 gene is found at positions 708-2693.

[0022]FIG. 2 is a genetic map of the H. somnus tbp Region. Restrictionsites are shown.

[0023]FIG. 3 shows the complete amino acid sequence of H. somnus Tbp1(SEQ ID NO:2).

[0024]FIG. 4 shows the complete amino acid sequence of H. somnus Tbp2(SEQ ID NO:3).

[0025]FIG. 5 shows Tbp1-specific serological response, measured asantibody titers, to vaccines containing recombinantly produced H. somnustransferrin-binding proteins. Bleed 1 was done preimmunization; Bleed 2was taken at the time of boost; and Bleed 3 was done prior to challenge.

[0026]FIG. 6 shows Tbp2-specific serological response, measured asantibody titers, to vaccines containing recombinantly produced H. somnustransferrin-binding proteins. Bleed 1 was done preimmunization; Bleed 2was taken at the time of boost; and Bleed 3 was done prior to challenge.

[0027]FIG. 7 shows mortality in groups of animals administered vaccinescontaining the recombinantly produced H. somnus transferrin-bindingproteins.

[0028]FIG. 8 depicts mean temperature obtained from animals administeredvaccines containing recombinantly produced H. somnus transferrin-bindingproteins and animals given placebos, as described in the examples.Results following H. somnus challenge are shown.

[0029]FIG. 9 shows depression scores from animals administered vaccinescontaining recombinantly produced H. somnus transferrin-binding proteinsand animals given placebos, as described in the examples. Scores fromDays 5-8, post H. somnus challenge, are shown.

[0030]FIG. 10 shows mean sick scores from animals administered vaccinescontaining recombinantly produced H. somnus transferrin-binding proteinsand animals given placebos, as described in the examples. Scores fromDays 5-8, post H. somnus challenge, are shown.

[0031] FIGS. 11A-11C depict a chromosomal fragment (SEQ ID NO:4) whichincludes the H. somnus lppB gene, occurring at positions 872-1906 of thefigure, and shows the corresponding LppB amino acid sequence (SEQ IDNO:5).

[0032]FIG. 12 depicts the Hopp/Woods antigenicity profile of H. somnusmature Tbp1.

[0033]FIG. 13 depicts the Kyte-Doolittle hydropathy plot (bottom offigure) and Argos transmembrane helices (top of figure) of H. somnusmature Tbp1.

[0034]FIG. 14 depicts the Hopp/Woods antigenicity profile of H. somnusTbp2.

[0035]FIG. 15 depicts the Kyte-Doolittle hydropathy plot of H. somnusTbp2.

DETAILED DESCRIPTION

[0036] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA technology, and immunology, which arewithin the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Vols. I, II and III, Second Edition (1989); Perbal,B., A Practical Guide to Molecular Cloning (1984); the series, MethodsIn Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C.C. Blackwell eds., 1986, Blackwell Scientific Publications).

[0037] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0038] The following amino acid abbreviations are used throughout thetext: Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Asparticacid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu(E) Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu(L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline:Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine:Tyr (Y) Valine: Val (V)

[0039] A. Definitions

[0040] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0041] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “an H. somnus transferrin binding protein”includes a mixture of two or more such proteins, and the like.

[0042] The terms “transferrin-binding protein”, “TF-binding protein” and“Tbp” (used interchangeably herein) or a nucleotide sequence encodingthe same, intends a protein or a nucleotide sequence, respectively,which is derived from an H. somnus tbp gene. The nucleotide sequence oftwo representative H. somnus tbp genes, termed “tbp1” and “tbp2” herein,and the corresponding amino acid sequence of the Tbp proteins encoded bythese gene, are depicted in the Figures. In particular, FIGS. 1A-1B (SEQID NO:1) show the nucleotide sequence of full-length tbp1 (occurring atnucleotide positions 2891-5803, inclusive) and tbp2 (occurring atnucleotide positions 708-2693, inclusive) and FIGS. 3 (SEQ ID NO:2) and4 (SEQ ID NO:3), show the full-length amino acid sequences of Tbp1 andTbp2, respectively. However, an H. somnus transferrin-binding protein asdefined herein is not limited to the depicted sequences as severalsubtypes of H. somnus are known and variations in transferrin-bindingproteins will occur between strains of H. somnus.

[0043] Furthermore, the derived protein or nucleotide sequences need notbe physically derived from the gene described above, but may begenerated in any manner, including for example, chemical synthesis,isolation (e.g., from H. somnus) or by recombinant production, based onthe information provided herein. Additionally, the term intends proteinshaving amino acid sequences substantially homologous (as defined below)to contiguous amino acid sequences encoded by the genes, which displayimmunological and/or transferrin-binding activity.

[0044] Thus, the terms intend full-length, as well as immunogenic,truncated and partial sequences, and active analogs and precursor formsof the proteins. Also included in the term are nucleotide fragments ofthe gene that include at least about 8 contiguous base pairs, morepreferably at least about 10-20 contiguous base pairs, and mostpreferably at least about 25 to 50, or more, contiguous base pairs ofthe gene. Such fragments are useful as probes and in diagnostic methods,discussed more fully below.

[0045] The terms also include those forms possessing, as well aslacking, the signal sequence, as well as the nucleic acid sequencescoding therefor. Additionally, the term intends forms of thetransferrin-binding proteins which lack the membrane anchor region, andnucleic acid sequences encoding proteins with such deletions. Suchdeletions may be desirable in systems that do not provide for secretionof the protein. Furthermore, the transferrin-binding domains of theproteins, may or may not be present. Thus, for example, if thetransferrin-binding protein will be used to purify transferrin, thetransferrin-binding domain will generally be retained. If the protein isto be used in vaccine compositions, immunogenic epitopes which may ormay not include the transferrin-binding domain, will be present.

[0046] The terms also include proteins in neutral form or in the form ofbasic or acid addition salts depending on the mode of preparation. Suchacid addition salts may involve free amino groups and basic salts may beformed with free carboxyls. Pharmaceutically acceptable basic and acidaddition salts are discussed further below. In addition, the proteinsmay be modified by combination with other biological materials such aslipids (both those occurring naturally with the molecule or other lipidsthat do not destroy immunological activity) and saccharides, or by sidechain modification, such as acetylation of amino groups, phosphorylationof hydroxyl side chains, oxidation of sulfhydryl groups, glycosylationof amino acid residues, as well as other modifications of the encodedprimary sequence.

[0047] The proteins of the present invention are normally found inassociation with lipid moieties. It is likely that the fatty acid moietypresent is a palmitic acid derivative. The antigens of the presentinvention, even though carrying epitopes derived from lipoproteins, donot require the presence of the lipid moiety. Furthermore, if the lipidis present, it need not be a lipid commonly associated with thelipoprotein, so long as the appropriate immunologic response iselicited. In any event, suitable fatty acids, such as but not limitedto, palmitic acid or palmitic acid analogs, can be conveniently added tothe desired amino acid sequence during synthesis, using standardtechniques. For example, palmitoyl bound to S-glyceryl-L-Cys (Pam₃-Cys)is commercially available (e.g. through Boehringer Mannheim, Dorval,Quebec) and can easily be incorporated into an amino acid sequenceduring synthesis. See, e.g. Deres et al. (1989) Nature 342:561. This isa particularly convenient method for production when relatively shortamino acid sequences are used. Similarly, recombinant systems can beused which will process the expressed proteins by adding suitable fattyacids. Representative systems for recombinant production are discussedfurther below.

[0048] The term therefore intends deletions, additions and substitutionsto the sequence, so long as the polypeptide functions to produce animmunological response as defined herein. In this regard, particularlypreferred substitutions will generally be conservative in nature, i.e.,those substitutions that take place within a family of amino acids. Forexample, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, or viceversa; an aspartate with a glutamate or vice versa; a threonine with aserine or vice versa; or a similar conservative replacement of an aminoacid with a structurally related amino acid, will not have a majoreffect on the biological activity. Proteins having substantially thesame amino acid sequence as the reference molecule, but possessing minoramino acid substitutions that do not substantially affect theimmunogenicity of the protein, are therefore within the definition ofthe reference polypeptide.

[0049] For example, the polypeptide of interest may include up to about5-10 conservative or non-conservative amino acid substitutions, or evenup to about 15-25 conservative or non-conservative amino acidsubstitutions, so long as the desired function of the molecule remainsintact. In this regard, substitutions occurring in the transmembranebinding domain and the signal sequence normally will not affectimmunogenicity. One of skill in the art may readily determine otherregions of the molecule of interest that can tolerate change byreference to the Hopp/Woods and Kyte-Doolittle plots shown in FIGS.12-15 herein.

[0050] An “isolated” nucleic acid molecule is a nucleic acid moleculeseparate and discrete from the whole organism with which the molecule isfound in nature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences (as defined below)in association therewith.

[0051] By “subunit vaccine composition” is meant a compositioncontaining at least one immunogenic polypeptide, but not all antigens,derived from or homologous to an antigen from a pathogen of interest.Such a composition is substantially free of intact pathogen cells orparticles, or the lysate of such cells or particles. Thus, a “subunitvaccine composition” is prepared from at least partially purified(preferably substantially purified) immunogenic polypeptides from thepathogen, or recombinant analogs thereof. A subunit vaccine compositioncan comprise the subunit antigen or antigens of interest substantiallyfree of other antigens or polypeptides from the pathogen.

[0052] The term “epitope” refers to the site on an antigen or hapten towhich specific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite.” Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

[0053] An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or protective immunological response such thatresistance of the mammary gland to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host and/or a quicker recovery time.

[0054] The terms “immunogenic” protein or polypeptide refer to an aminoacid sequence which elicits an immunological response as describedabove. An “immunogenic” protein or polypeptide, as used herein, includesthe full-length sequence of the transferrin-binding protein in question,with or without the signal sequence, membrane anchor domain and/ortransferrin-binding domain, analogs thereof, or immunogenic fragmentsthereof. By “immunogenic fragment” is meant a fragment of atransferrin-binding protein which includes one or more epitopes and thuselicits the immunological response described above. Such fragments canbe identified using any number of epitope mapping techniques, well knownin the art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots. FIGS. 12-14 herein depict Hopp/Woods andKyte-Doolittle profiles for representative proteins encompassed by theinvention.

[0055] Immunogenic fragments, for purposes of the present invention,will usually include at least about 3 amino acids, preferably at leastabout 5 amino acids, more preferably at least about 10-15 amino acids,and most preferably 25 or more amino acids, of the parenttransferrin-binding protein molecule. There is no critical upper limitto the length of the fragment, which may comprise nearly the full-lengthof the protein sequence, or even a fusion protein comprising two or moreepitopes of Tbp1 and/or Tbp2.

[0056] “Native” proteins or polypeptides refer to proteins orpolypeptides isolated from the source in which the proteins naturallyoccur. “Recombinant” polypeptides refer to polypeptides produced byrecombinant DNA techniques; i.e., produced from cells transformed by anexogenous DNA construct encoding the desired polypeptide. “Synthetic”polypeptides are those prepared by chemical synthesis.

[0057] A “vector” is a replicon, such as a plasmid, phage, or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment.

[0058] A DNA “coding sequence” or a “nucleotide sequence encoding” aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide in vitro or in vivo when placed under thecontrol of appropriate regulatory elements. The boundaries of the codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, procaryotic sequences, cDNA fromeucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian)DNA, and even synthetic DNA sequences. A transcription terminationsequence will usually be located 3′ to the coding sequence.

[0059] DNA “control elements” refers collectively to promoters, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, whichcollectively provide for the transcription and translation of a codingsequence in a host cell. Not all of these control sequences need alwaysbe present in a recombinant vector so long as the desired gene iscapable of being transcribed and translated.

[0060] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter and the coding sequence and the promoter canstill be considered “operably linked” to the coding sequence.

[0061] A control element, such as a promoter, “directs thetranscription” of a coding sequence in a cell when RNA polymerase willbind the promoter and transcribe the coding sequence into mRNA, which isthen translated into the polypeptide encoded by the coding sequence.

[0062] A “host cell” is a cell which has been transformed, or is capableof transformation, by an exogenous nucleic acid molecule.

[0063] A cell has been “transformed” by exogenous DNA when suchexogenous DNA has been introduced inside the cell membrane. ExogenousDNA may or may not be integrated (covalently linked) into chromosomalDNA making up the genome of the cell. In procaryotes and yeasts, forexample, the exogenous DNA may be maintained on an episomal element,such as a plasmid. With respect to eucaryotic cells, a stablytransformed cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

[0064] “Homology” refers to the percent identity between twopolynucleotide or two polypeptide moieties. Two DNA, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 80%-85%, preferably at least about 90%,and most preferably at least about 95%-98% sequence identity over adefined length of the molecules. As used herein, substantiallyhomologous also refers to sequences showing complete identity to thespecified DNA or polypeptide sequence.

[0065] In general, “identity” refers to an exactnucleotide-to-nucleotide or amino acid-to-amino acid correspondence oftwo polynucleotides or polypeptide sequences, respectively. Percentidentity can be determined by a direct comparison of the sequenceinformation between two molecules by aligning the sequences, countingthe exact number of matches between the two aligned sequences, dividingby the length of the shorter sequence, and multiplying the result by100. Readily available computer programs can be used to aid in theanalysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence andStructure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedicalResearch Foundation, Washington, D.C., which adapts the local homologyalgorithm of Smith and Waterman (1981) Advances in Appl. Math. 2:482-489for peptide analysis. Programs for determining nucleotide sequenceidentity are available in the Wisconsin Sequence Analysis Package,Version 8 (available from Genetics Computer Group, Madison, Wis.) forexample, the BESTFIT, FASTA and GAP programs, which also rely on theSmith and Waterman algorithm. These programs are readily utilized withthe default parameters recommended by the manufacturer and described inthe Wisconsin Sequence Analysis Package referred to above. For example,percent identity of a particular nucleotide sequence to a referencesequence can be determined using the homology algorithm of Smith andWaterman with a default scoring table and a gap penalty of sixnucleotide positions.

[0066] Another method of establishing percent identity in the context ofthe present invention is to use the MPSRCH package of programscopyrighted by the University of Edinburgh, developed by John F. Collinsand Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (MountainView, Calif.). From this suite of packages the Smith-Waterman algorithmcan be employed where default parameters are used for the scoring table(for example, gap open penalty of 12, gap extension penalty of one, anda gap of six). From the data generated the “Match” value reflects“sequence identity.” Other suitable programs for calculating the percentidentity or similarity between sequences are generally known in the art,for example, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0067] Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

[0068] By the term “degenerate variant” is intended a polynucleotidecontaining changes in the nucleic acid sequence thereof, that encodes apolypeptide having the same amino acid sequence as the polypeptideencoded by the polynucleotide from which the degenerate variant isderived.

[0069] The term “functionally equivalent” intends that the amino acidsequence of a transferrin-binding protein is one that will elicit asubstantially equivalent or enhanced immunological response, as definedabove, as compared to the response elicited by a transferrin-bindingprotein having identity with the reference transferrin-binding protein,or an immunogenic portion thereof.

[0070] A “heterologous” region of a DNA construct is an identifiablesegment of DNA within or attached to another DNA molecule that is notfound in association with the other molecule in nature. Thus, when theheterologous region encodes a bacterial gene, the gene will usually beflanked by DNA that does not flank the bacterial gene in the genome ofthe source bacteria. Another example of the heterologous coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., synthetic sequences having codons different from the nativegene). Allelic variation or naturally occurring mutational events do notgive rise to a heterologous region of DNA, as used herein.

[0071] The term “treatment” as used herein refers to either (i) theprevention of infection or reinfection (prophylaxis), or (ii) thereduction or elimination of symptoms of the disease of interest(therapy).

[0072] As used herein, a “biological sample” refers to a sample oftissue or fluid isolated from a subject, including but not limited to,for example, blood, plasma, serum, fecal matter, urine, bone marrow,bile, spinal fluid, lymph fluid, samples of the skin, externalsecretions of the skin, respiratory, intestinal, and genitourinarytracts, tears, saliva, milk, blood cells, organs, biopsies and alsosamples of in vitro cell culture constituents including but not limitedto conditioned media resulting from the growth of cells and tissues inculture medium, e.g., recombinant cells, and cell components.

[0073] As used herein, the terms “label” and “detectable label” refer toa molecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.The term “fluorescer” refers to a substance or a portion thereof whichis capable of exhibiting fluorescence in the detectable range.Particular examples of labels which may be used under the inventioninclude fluorescein, rhodamine, dansyl, umbelliferone, Texas red,luminol, NADPH and α-β-galactosidase.

[0074] D. General Methods

[0075] Central to the present invention is the discovery of genesencoding two H. somnus transferrin-binding proteins, termed “Tbp1” and“Tbp2,” respectively herein. In particular, the genes for H. somnustransferrin-binding protein 1 (“tbp1”) and H. somnus transferrin-bindingprotein 2 (“tbp2”) have been isolated, sequenced and characterized, andthe protein sequences for Tbp1 and Tbp2 deduced therefrom. The completeDNA sequences are shown in FIGS. 1A-1B and the protein sequences forTbp1 and Tbp2 shown in FIGS. 3 (SEQ ID NO:2) and 4 (SEQ ID NO:3),respectively.

[0076] As described in the examples, full-length tbp1, depicted atnucleotide positions 2891-5803, inclusive, of FIGS. 1A-1B, encodes afull-length Tbp1 protein of approximately 971 amino acids, shown asamino acids 1-971, inclusive, of FIG. 3 (SEQ ID NO:2). The protein has apredicted molecular weight of about 109,725 kDa. The full-lengthsequence includes a signal peptide of 28 amino acids, occurring atpositions 1 to 28 of FIG. 3. Thus, the mature Tbp1 sequence isrepresented by amino acids 29 to 971, inclusive, of FIG. 3 and isencoded by the nucleotide sequence depicted at positions 2975 to 5803,inclusive of FIGS. 1A-1B. FIG. 12 shows the Hopp/Woods antigenicityprofile of H. somnus mature Tbp1. FIG. 13 depicts the Kyte-Doolittlehydropathy plot (bottom of figure) and Argos transmembrane helices (topof figure) of H. somnus mature Tbp1.

[0077] Full-length tbp2, depicted at nucleotide positions 708-2693,inclusive, of FIGS. 1A-1C (SEQ ID NO:1), encodes a full-length Tbp2protein of approximately 662 amino acids, shown as amino acids 1-662,inclusive, of FIG. 4 (SEQ ID NO:3). The protein has a predictedmolecular weight of about 71,311 kDa. The full-length sequence includesa signal peptide of 19 amino acids, occurring at positions 1 to 19 ofFIG. 4. Thus, the mature Tbp2 sequence is represented by amino acids 20to 662, inclusive, of FIG. 4 and is encoded by the nucleotide sequencedepicted at positions 765 to 2683, of FIGS. 1A-1B. FIG. 14 depicts theHopp/Woods antigenicity profile of H. somnus Tbp2 and FIG. 15 depictsthe Kyte-Doolittle hydropathy plot of H. somnus Tbp2. Unlike Tbp1, notransmembrane binding domains are present in the Tbp2 molecule.

[0078] The H. somnus transferrin-binding proteins, immunogenic fragmentsthereof or chimeric proteins including one or more epitopes of Tbp1 andTbp2, can be provided, either alone or in combination, in subunitvaccine compositions to treat or prevent bacterial infections caused byH. somnus, including, but not limited to, hemophilosis, thromboembolicmeningoencephalitis (ITEME), septicemia, arthritis, and pneumonia(Corbeill, L. B., Can. J. Vet. Res. (1990) 54:S57-S62; Harris, F. W.,and Janzen, E. D., Can. Vet. J. (1990) 30:816-822; Humphrey, J. D., andStephens, L. R., Vet. Bull. (1983) 53:987-1004), as well as myocarditis,pericarditis, spontaneous abortion, infertility and mastitis.

[0079] In addition to use in vaccine compositions, the proteins andfragments thereof, antibodies thereto, and genes coding therefor, can beused as diagnostic reagents to detect the presence of infection in amammalian subject. Similarly, the genes encoding the proteins can becloned and used to design probes to detect and isolate homologous genesin other bacterial strains. For example, fragments comprising at leastabout 15-20 nucleotides, more preferably at least about 20-50nucleotides, and most preferably about 60-100 or more nucleotides, willfind use in these embodiments. The H. somnus transferrin-bindingproteins also find use in purifying transferring from Haemophilusspecies and from recombinant host cells expressing the same.

[0080]H. somnus transferrin binding proteins can be used in vaccinecompositions either alone or in combination with other bacterial,fungal, viral or protozoal antigens. These antigens can be providedseparately or even as fusion proteins comprising one or more epitopes ofthe transferrin-binding proteins fused together and/or to one or more ofthe above antigens.

[0081] For example, other immunogenic proteins from H. somnus can beused in the subject vaccines, including, but not limited to, H. somnusLppA, LppB and/or LppC polypeptides, H. somnus haemin-binding protein,and H. somnus haemolysin. All of these H. somnus proteins are describedin International Publication No. WO 93/21323, published Oct. 28, 1993).For example, FIGS. 11A-11C depict the H. somnus LppB protein (SEQ IDNO:5) and the gene coding therefor (positions 872-1906 of SEQ ID NO:4).The H. somnus LppB preprotein is encoded by nucleotide positions 872through 1906 (amino acid residues 1 through 345) and the mature proteinis encoded by nucleotide positions 920 through 1906 (amino acid residues17 through 345). The entire LppB protein, or fragments comprisingimmunogenic polypeptides of the protein, can be used in vaccinecompositions in combination with either or both of the H. somnustransferrin binding proteins.

[0082] Production of Transferrin-binding Proteins

[0083] The above described transferrin-binding proteins and activefragments, analogs and chimeric proteins derived from the same, can beproduced by a variety of methods. Specifically, transferrin-bindingproteins can be isolated directly from bacteria which express the same.The proteins can be isolated directly from H. somnus from outer membranepreparations, using standard purification techniques. See, e.g. Theisenand Potter (1992) Infect. Immun. 60:826-831. The desired proteins canthen be further purified i.e. by column chromatography, HPLC,immunoadsorbent techniques or other conventional methods well known inthe art.

[0084] Alternatively, the proteins can be recombinantly produced asdescribed herein. As explained above, these recombinant products cantake the form of partial protein sequences, full-length sequences,precursor forms that include signal sequences, mature forms withoutsignals, or even fusion proteins (e.g., with an appropriate leader forthe recombinant host, or with another subunit antigen sequence for H.somnus or another pathogen).

[0085] The tbp genes of the present invention can be isolated based onthe ability of the protein products to bind transferrin, usingtransferrin-binding assays as described below. Thus, gene libraries canbe constructed and the resulting clones used to transform an appropriatehost cell. Colonies can be pooled and screened for clones havingtransferrin-binding activity. Colonies can also be screened usingpolyclonal serum or monoclonal antibodies to the transferrin-bindingprotein.

[0086] Alternatively, once the amino acid sequences are determined,oligonucleotide probes which contain the codons for a portion of thedetermined amino acid sequences can be prepared and used to screengenomic or cDNA libraries for genes encoding the subject proteins. Thebasic strategies for preparing oligonucleotide probes and DNA libraries,as well as their screening by nucleic acid hybridization, are well knownto those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I,supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,supra; Sambrook et al., supra. Once a clone from the screened libraryhas been identified by positive hybridization, it can be confirmed byrestriction enzyme analysis and DNA sequencing that the particularlibrary insert contains a transferrin-binding protein gene or a homologthereof. The genes can then be further isolated using standardtechniques and, if desired, PCR approaches or restriction enzymesemployed to delete portions of the full-length sequence.

[0087] Similarly, genes can be isolated directly from bacteria usingknown techniques, such as phenol extraction and the sequence furthermanipulated to produce any desired alterations. See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolateDNA.

[0088] Alternatively, DNA sequences encoding the proteins of interestcan be prepared synthetically rather than cloned. The DNA sequences canbe designed with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

[0089] Once coding sequences for the desired proteins have been preparedor isolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra.

[0090] The gene can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. If a signal sequence is included, it caneither be the native, homologous sequence, or a heterologous sequence.For example, the signal sequence for the particular H. somnustransferrin-binding protein, can be used for secretion thereof, as can anumber of other signal sequences, well known in the art. Leadersequences can be removed by the host in post-translational processing.See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

[0091] Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

[0092] The control sequences and other regulatory sequences may beligated to the coding sequence prior to insertion into a vector, such asthe cloning vectors described above. Alternatively, the coding sequencecan be cloned directly into an expression vector which already containsthe control sequences and an appropriate restriction site.

[0093] In some cases it may be necessary to modify the coding sequenceso that it may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the transferrin-bindingprotein. Mutants or analogs may be prepared by the deletion of a portionof the sequence encoding the protein, by insertion of a sequence, and/orby substitution of one or more nucleotides within the sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, are described in, e.g., Sambrook et al., supra; DNACloning, supra; Nucleic Acid Hybridization, supra.

[0094] The expression vector is then used to transform an appropriatehost cell. A number of mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0095] Depending on the expression system and host selected, theproteins of the present invention are produced by culturing host cellstransformed by an expression vector described above under conditionswhereby the protein of interest is expressed. The protein is thenisolated from the host cells and purified. If the expression systemsecretes the protein into the growth media, the protein can be purifieddirectly from the media. If the protein is not secreted, it is isolatedfrom cell lysates. The selection of the appropriate growth conditionsand recovery methods are within the skill of the art.

[0096] The proteins of the present invention may also be produced bychemical synthesis such as solid phase peptide synthesis, using knownamino acid sequences or amino acid sequences derived from the DNAsequence of the genes of interest. Such methods are known to thoseskilled in the art. See, e.g., J. M. Stewart and J. D. Young, SolidPhase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill.(1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis,Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, AcademicPress, New York, (1980), pp. 3-254, for solid phase peptide synthesistechniques; and M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classicalsolution synthesis. Chemical synthesis of peptides may be preferable ifa small fragment of the antigen in question is capable of raising animmunological response in the subject of interest.

[0097] The transferrin-binding proteins of the present invention, ortheir fragments, can be used to produce antibodies, both polyclonal andmonoclonal. If polyclonal antibodies are desired, a selected mammal,(e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen ofthe present invention, or its fragment, or a mutated antigen. Serum fromthe immunized animal is collected and treated according to knownprocedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370. Ifserum containing polyclonal antibodies is used, the polyclonalantibodies can be purified by immunoaffinity chromatography, using knownprocedures.

[0098] Monoclonal antibodies to the transferrin-binding proteins and tothe fragments thereof, can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byusing hybridoma technology is well known. Immortal antibody-producingcell lines can be created by cell fusion, and also by other techniquessuch as direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. See, e.g., M. Schreier et al.,Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodiesand T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies(1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890.Panels of monoclonal antibodies produced against the transferrin-bindingproteins, or fragments thereof, can be screened for various properties;i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies areuseful in purification, using immunoaffinity techniques, of theindividual antigens which they are directed against. Both polyclonal andmonoclonal antibodies can also be used for passive immunization or canbe combined with subunit vaccine preparations to enhance the immuneresponse. Polyclonal and monoclonal antibodies are also useful fordiagnostic purposes.

[0099] Vaccine Formulations and Administration

[0100] The transferrin-binding proteins of the present invention can beformulated into vaccine compositions, either alone, in combinationand/or with other antigens, for use in immunizing subjects as describedbelow. Methods of preparing such formulations are described in, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 18 Edition, 1990. Typically, the vaccines of the present inventionare prepared as injectables, either as liquid solutions or suspensions.Solid forms suitable for solution in or suspension in liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.The active immunogenic ingredient is generally mixed with a compatiblepharmaceutical vehicle, such as, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents and pH bufferingagents.

[0101] Adjuvants which enhance the effectiveness of the vaccine may alsobe added to the formulation. Adjuvants may include for example, muramyldipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammoniumbromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, andother substances known in the art.

[0102] The transferrin-binding proteins may be linked to a carrier inorder to increase the immunogenicity thereof. Suitable carriers includelarge, slowly metabolized macromolecules such as proteins, includingserum albumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art; polysaccharides, such as sepharose, agarose, cellulose,cellulose beads and the like; polymeric amino acids such as polyglutamicacid, polylysine, and the like; amino acid copolymers; and inactivevirus particles.

[0103] The transferrin-binding proteins may be used in their native formor their functional group content may be modified by, for example,succinylation of lysine residues or reaction with Cys-thiolactone. Asulfhydryl group may also be incorporated into the carrier (or antigen)by, for example, reaction of amino functions with 2-iminothiolane or theN-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitablecarriers may also be modified to incorporate spacer arms (such ashexamethylene diamine or other bifunctional molecules of similar size)for attachment of peptides.

[0104] Other suitable carriers for the transferrin-binding proteins ofthe present invention include VP6 polypeptides of rotaviruses, orfunctional fragments thereof, as disclosed in U.S. Pat. No. 5,071,651,incorporated herein by reference. Also useful is a fusion product of aviral protein and the subject immunogens made by methods disclosed inU.S. Pat. No. 4,722,840. Still other suitable carriers include cells,such as lymphocytes, since presentation in this form mimics the naturalmode of presentation in the subject, which gives rise to the immunizedstate. Alternatively, the proteins of the present invention may becoupled to erythrocytes, preferably the subject's own erythrocytes.Methods of coupling peptides to proteins or cells are known to those ofskill in the art.

[0105] Furthermore, the transferrin-binding proteins (or complexesthereof) may be formulated into vaccine compositions in either neutralor salt forms. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the activepolypeptides) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from freecarboxyl groups may also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0106] Vaccine formulations will contain a “therapeutically effectiveamount” of the active ingredient, that is, an amount capable ofeliciting an immune response in a subject to which the composition isadministered. In the treatment and prevention of H. somnus infection,for example, a “therapeutically effective amount” would preferably be anamount that enhances resistance of the mammal in question to newinfection and/or reduces the clinical severity of the disease. Suchprotection will be demonstrated by either a reduction or lack ofsymptoms normally displayed by an infected host and/or a quickerrecovery time.

[0107] The exact amount is readily determined by one skilled in the artusing standard tests. The transferrin-binding protein concentration willtypically range from about 1% to about 95% (w/w) of the composition, oreven higher or lower if appropriate. With the present vaccineformulations, 5 to 500 μg of active ingredient per ml of injectedsolution, preferably 10 to 100 μg of active ingredient per ml, should beadequate to raise an immunological response when a dose of 1 to 3 ml peranimal is administered.

[0108] To immunize a subject, the vaccine is generally administeredparenterally, usually by intramuscular injection. Other modes ofadministration, however, such as subcutaneous, intraperitoneal andintravenous injection, are also acceptable. The quantity to beadministered depends on the animal to be treated, the capacity of theanimal's immune system to synthesize antibodies, and the degree ofprotection desired. Effective dosages can be readily established by oneof ordinary skill in the art through routine trials establishing doseresponse curves. The subject is immunized by administration of thevaccine in at least one dose, and preferably two doses. Moreover, theanimal may be administered as many doses as is required to maintain astate of immunity to infection.

[0109] Additional vaccine formulations which are suitable for othermodes of administration include suppositories and, in some cases,aerosol, intranasal, oral formulations, and sustained releaseformulations. For suppositories, the vehicle composition will includetraditional binders and carriers, such as, polyalkaline glycols, ortriglycerides. Such suppositories may be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10% (w/w),preferably about 1% to about 2%. Oral vehicles include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium, stearate, sodium saccharin cellulose,magnesium carbonate, and the like. These oral vaccine compositions maybe taken in the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations, or powders, and contain fromabout 10% to about 95% of the active ingredient, preferably about 25% toabout 70%.

[0110] Intranasal formulations will usually include vehicles thatneither cause irritation to the nasal mucosa nor significantly disturbciliary function. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

[0111] Controlled or sustained release formulations are made byincorporating the protein into carriers or vehicles such as liposomes,nonresorbable impermeable polymers such as ethylenevinyl acetatecopolymers and Hytrel® copolymers, swellable polymers such as hydrogels,or resorbable polymers such as collagen and certain polyacids orpolyesters such as those used to make resorbable sutures. Thetransferrin-binding proteins can also be delivered using implantedmini-pumps, well known in the art.

[0112] The transferrin-binding proteins of the instant invention canalso be administered via a carrier virus which expresses the same.Carrier viruses which will find use with the instant invention includebut are not limited to the vaccinia and other pox viruses, adenovirus,and herpes virus. By way of example, vaccinia virus recombinantsexpressing the novel proteins can be constructed as follows. The DNAencoding the particular protein is first inserted into an appropriatevector so that it is adjacent to a vaccinia promoter and flankingvaccinia DNA sequences, such as the sequence encoding thymidine kinase(TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the instantprotein into the viral genome. The resulting TK-recombinant can beselected by culturing the cells in the presence of 5-bromodeoxyuridineand picking viral plaques resistant thereto.

[0113] An alternative route of administration involves gene therapy ornucleic acid immunization. Thus, nucleotide sequences (and accompanyingregulatory elements) encoding the subject transferrin-binding proteinscan be administered directly to a subject for in vivo translationthereof. Alternatively, gene transfer can be accomplished bytransfecting the subject's cells or tissues ex vivo and reintroducingthe transformed material into the host. DNA can be directly introducedinto the host organism, i.e., by injection (see U.S. Pat. Nos. 5,580,859and 5,589,466; International Publication No. WO/90/11092; and Wolff etal. (1990) Science 247:1465-1468). Liposome-mediated gene transfer canalso be accomplished using known methods. See, e.g., U.S. Pat. No.5,703,055; Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol.4:206-209; Brigham et al. (1989) Am. J. Med. Sci. 298:278-281; Canonicoet al. (1991) Clin. Res. 39:219A; and Nabel et al. (1990) Science249:1285-1288. Targeting agents, such as antibodies directed againstsurface antigens expressed on specific cell types, can be covalentlyconjugated to the liposomal surface so that the nucleic acid can bedelivered to specific tissues and cells susceptible to infection.

[0114] Diagnostic Assays

[0115] As explained above, the transferrin-binding proteins of thepresent invention may also be used as diagnostics to detect the presenceof reactive antibodies of H. somnus in a biological sample in order todetermine the presence of H. somnus infection. For example, the presenceof antibodies reactive with transferrin-binding proteins can be detectedusing standard electrophoretic and immunodiagnostic techniques,including immunoassays such as competition, direct reaction, or sandwichtype assays. Such assays include, but are not limited to, Western blots;agglutination tests; enzyme-labeled and mediated immunoassays, such asELISAs; biotin/avidin type assays; radioimmunoassays;immunoelectrophoresis; immunoprecipitation, etc. The reactions generallyinclude revealing labels such as fluorescent, chemiluminescent,radioactive, enzymatic labels or dye molecules, or other methods fordetecting the formation of a complex between the antigen and theantibody or antibodies reacted therewith.

[0116] The aforementioned assays generally involve separation of unboundantibody in a liquid phase from a solid phase support to whichantigen-antibody complexes are bound. Solid supports which can be usedin the practice of the invention include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

[0117] Typically, a solid support is first reacted with a solid phasecomponent (e.g., one or more transferrin-binding proteins) undersuitable binding conditions such that the component is sufficientlyimmobilized to the support. Sometimes, immobilization of the antigen tothe support can be enhanced by first coupling the antigen to a proteinwith better binding properties. Suitable coupling proteins include, butare not limited to, macromolecules such as serum albumins includingbovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulinmolecules, thyroglobulin, ovalbumin, and other proteins well known tothose skilled in the art. Other molecules that can be used to bind theantigens to the support include polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andthe like. Such molecules and methods of coupling these molecules to theantigens, are well known to those of ordinary skill in the art. See,e.g., Brinkley, M. A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al.,J. Appl. Biochem. (1984) 6:56-63; and Anjaneyulu and Staros,International J. of Peptide and Protein Res. (1987) 30:117-124.

[0118] After reacting the solid support with the solid phase component,any non-immobilized solid-phase components are removed from the supportby washing, and the support-bound component is then contacted with abiological sample suspected of containing ligand moieties (e.g.,antibodies toward the immobilized antigens) under suitable bindingconditions. After washing to remove any non-bound ligand, a secondarybinder moiety is added under suitable binding conditions, wherein thesecondary binder is capable of associating selectively with the boundligand. The presence of the secondary binder can then be detected usingtechniques well known in the art.

[0119] More particularly, an ELISA method can be used, wherein the wellsof a microtiter plate are coated with a transferrin-binding protein. Abiological sample containing or suspected of containinganti-transferrin-binding protein immunoglobulin molecules is then addedto the coated wells. After a period of incubation sufficient to allowantibody binding to the immobilized antigen, the plate(s) can be washedto remove unbound moieties and a detectably labeled secondary bindingmolecule added. The secondary binding molecule is allowed to react withany captured sample antibodies, the plate washed and the presence of thesecondary binding molecule detected using methods well known in the art.

[0120] Thus, in one particular embodiment, the presence of boundanti-transferrin-binding antigen ligands from a biological sample can bereadily detected using a secondary binder comprising an antibodydirected against the antibody ligands. A number of anti-bovineimmunoglobulin (Ig) molecules are known in the art which can be readilyconjugated to a detectable enzyme label, such as horseradish peroxidase,alkaline phosphatase or urease, using methods known to those of skill inthe art. An appropriate enzyme substrate is then used to generate adetectable signal. In other related embodiments, competitive-type ELISAtechniques can be practiced using methods known to those skilled in theart.

[0121] Assays can also be conducted in solution, such that thetransferrin-binding proteins and antibodies specific for those proteinsform complexes under precipitating conditions. In one particularembodiment, transferrin-binding proteins can be attached to a solidphase particle (e.g., an agarose bead or the like) using couplingtechniques known in the art, such as by direct chemical or indirectcoupling. The antigen-coated particle is then contacted under suitablebinding conditions with a biological sample suspected of containingantibodies for the transferrin-binding proteins. Cross-linking betweenbound antibodies causes the formation of particle-antigen-antibodycomplex aggregates which can be precipitated and separated from thesample using washing and/or centrifugation. The reaction mixture can beanalyzed to determine the presence or absence of antibody-antigencomplexes using any of a number of standard methods, such as thoseimmunodiagnostic methods described above.

[0122] In yet a further embodiment, an immunoaffinity matrix can beprovided, wherein a polyclonal population of antibodies from abiological sample suspected of containing anti-transferrin-bindingmolecules is immobilized to a substrate. In this regard, an initialaffinity purification of the sample can be carried out using immobilizedantigens. The resultant sample preparation will thus only containanti-H. soinus moieties, avoiding potential nonspecific bindingproperties in the affinity support. A number of methods of immobilizingimmunoglobulins (either intact or in specific fragments) at high yieldand good retention of antigen binding activity are known in the art. Notbeing limited by any particular method, immobilized protein A or proteinG can be used to immobilize immunoglobulins.

[0123] Accordingly, once the immunoglobulin molecules have beenimmobilized to provide an immunoaffinity matrix, labeledtransferrin-binding proteins are contacted with the bound antibodiesunder suitable binding conditions. After any non-specifically boundantigen has been washed from the immunoaffinity support, the presence ofbound antigen can be determined by assaying for label using methodsknown in the art.

[0124] Additionally, antibodies raised to the transferrin-bindingproteins, rather than the transferrin-binding proteins themselves, canbe used in the above-described assays in order to detect the presence ofantibodies to the proteins in a given sample. These assays are performedessentially as described above and are well known to those of skill inthe art.

[0125] The above-described assay reagents, including thetransferrin-binding proteins, or antibodies thereto, can be provided inkits, with suitable instructions and other necessary reagents, in orderto conduct immunoassays as described above. The kit can also contain,depending on the particular immunoassay used, suitable labels and otherpackaged reagents and materials (i.e. wash buffers and the like).Standard immunoassays, such as those described above, can be conductedusing these kits.

[0126] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0127] C. Experimental

EXAMPLE 1 Isolation and Cloning of H. somnus tbp1 and tbp2 Materials andMethods

[0128] Bacterial Strains, Plasmids and Growth Conditions.

[0129]E. coli DH5αF′IQ[φ80 lacZΔM15 endA1 recA1 hsdR17 (r_(K) ⁻ m_(K) ⁺)supE44 thi-1 λ⁻ gyrA96 re1A1 Δ(lacZYA-argF)U169/F′ lacI^(q) proAB⁺lacZΔM15 zzf::Tn5 (Km)] (available commercially from, e.g., Stratagene),and JM105 (Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Vols. I, II and III, Second Edition (1989)) were from thelaboratory collection. E. coli strains were grown aerobically at 37° C.in Luria-Bertani (LB) or in M63 defined medium containing 0.5% (vol/vol)glycerol supplemented with 2% (wt/vol) casamino acids. Ampicillin wasused at 50 μg/ml. H. somnus strain HS25 was obtained from the lung of acalf which died of pneumonia and has been shown to induce experimentalhemophilosis in calves. The conditions for the storage and growth of H.somnus have been described previously. Theisen and Potter (1992) J.Bacteriol. 174:17-23. Liquid cultures were made in brain heart infusionbroth (Difco laboratories, Detroit, Mich.) supplemented with 0.1%(wt/vol) Tris base and 0.001% (wt/vol) thiamine monophosphate (BHI-TT).Growth in iron-deplete conditions was obtained by adding the ironchelator 2,2′-dipyridyl at a final concentration of 300 μM to the BHI-TTmedium.

[0130] The expression library consisted of 2- to 7-kb partial Sau3A1restriction fragments of H. somnus genomic DNA ligated into the BamHIrestriction site of pGH432 (see, Theisen and Potter (1992) J. Bacteriol.174:17-23), allowing for in-frame fusions with an artificial leaderpeptide whose expression can be induced from a lacO controlled tacpromoter (Advanced Vectors, Hopkins, Minn.).

[0131] Preparation of the Native Transferrin Receptors of H. somnus andSpecific Antisera.

[0132] Transferrin-binding proteins (Tbp) from H. somnus strain HS25were isolated by affinity chromatography using bovine transferrin asdescribed by ogunnariwo et al. (1990) Microbiol. Path. 9:397-406.Briefly, total membranes of H. somnus were mixed with biotinylatedbovine transferrin before solubilization with EDTA-Sarkosyl and additionto streptavidin-agarose. The affinity bound material was released bywashing with various buffers. Specific antiserum against thetransferrin-binding proteins was raised in a rabbit by conventionalmethods.

[0133] PAGE and Immunoblotting.

[0134] SDS-polyacrylamide gel electrophoresis (PAGE) of proteins wasperformed using the method described by Laemmli (Laemmli, U.K. (1970)Nature 227:680-685). Immunoblotting was carried out using standardtechniques described by the manufacturer of the electroblot apparatus(BioRad Laboratories). The primary antiserum was rabbit serum raisedagainst H. somnus Tbp purified by affinity chromatography or bovinehyperimmune serum raised against live H. somnus HS25 (Theisen and Potter(1992) J. Bacteriol. 174:17-23). The seroreactive proteins were detectedwith goat anti-rabbit immunoglobulin G coupled to alkaline phosphatase(PhoA) or with goat anti-bovine immunoglobulin G coupled to PhoA(Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.). PhoAactivity was visualized using the nitroblue tetrazolium-BCIP system(Promega, Madison, Wis.).

[0135] Colony Immunoblot of an H. somnus Genomic Library.

[0136] JM105 cells harboring the plasmid expression library of H. somnusHS25 were streaked on agar plates and tested for the production of Tbpby the colony blot method (French et al. (1986) Anal. Biochem.156:417-423) using rabbit serum raised against affnity-purified H.somnus Tbp.

[0137] DNA Techniques.

[0138] Standard methods were used for DNA manipulations (Sambrook,supra). The DNA restriction enzyme digests were done in T4 DNApolymerase buffer (Sambrook, supra) with 1 mM dithiothreitol andsupplemented with 3 mM spermidine. All synthetic oligonucleotides wereproduced with a Gene Assembler Plus (Pharmacia LKB Biotechnology,Uppsala, Sweden) DNA synthesizer. DNA sequencing was performed by thedideoxy chain-termination method (Sanger et al. (1977) Proc. Natl. Acad.Sci. USA 74:5463-5467; T7 sequencing kit (Pharmacia)) on single strandedDNA derived from nested deletions prepared by exonuclease III treatment(Henikoff, S. (1977) Gene 28:351-359; double-stranded nested deletionkit (Pharmacia)) or double stranded DNA as template. Sequences wereanalysed with the PCGENE software package (IntelliGenetics, MountainView, Calif.).

[0139] Inverse PCR, based on the method of Ochman et al. (Ochman et al.(1990) “Amplification of flanking sequences by inverse PCR.” in PCRProtocols: A Guide to Methods and Applications. Academic Press) was usedfor cloning tbp2 from H.somnus HS25.

[0140] Enrichment of Recombinantly Produced Tbp1 and Tbp2 from E. coli

[0141] For Tbp1, Bacteria were grown to mid-log phase in one liter ofL-broth supplemented with 50 μg/ml of ampicillin. When the absorbance at600 nm reached 0.6, isopropyl-β,D-thiogalactoside (IPTG) was added to afinal concentration of 1 mM and the cultures were incubated withvigorous agitation for 2 h at 37° C. The bacteria were harvested bycentrifugation, resuspended in 40 ml of 25% sucrose/50 mM Tris-HClbuffer (pH 8) and frozen at −70° C. The frozen cells were thawed at roomtemperature and 10 ml of lysozyme (10 mg/ml in 250 mM Tris-HCl, pH 8)was added. After 15 minutes on ice, 300 ml of detergent mix (5 parts of20 mM Tris-HCl, pH 7.4/300 mM sodium chloride/2% deoxycholic acid/2%Nonidet-P40 and 4 parts of 100 mM Tris-HCl, pH 8/50 mM EDTA/2% TritonX-100) were added. The viscosity was reduced by sonication and proteinaggregates were harvested by centrifugation at 27,000× g for 15 minutes.The pellets were dissolved in a minimal volume of 4 M guanidinehydrochloride. The proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the protein concentrationwas estimated by comparing the intensity of the Coomassie blue-stainedbands to a bovine serum albumin standard.

[0142] Tbp2 was purified from total outer membranes with Sarkosyl.Briefly, bacteria were grown to mid-log phase in one liter of L-brothsupplemented with ampicillin. When the absorbance at 600 nm reachedapproximately 0.6, IPTG was added to a final concentration of 1 mM andthe cultures were incubated with vigorous agitation for 2-4 h at 37° C.The bacteria were harvested by centrifugation, resuspended in Tris-EDTAbuffer, pH 8, and treated with lysozyme as described above. Cells weredisrupted by sonication and insoluble cell debris was removed bycentrifugation. The supernatant was then layered on a sucrose gradientand the outer membrane protein band withdrawn with a syringe followingovernight centrifugation. Following dialysis, lipoproteins includingTbp2, were selectively solubilized by mixing the membrane fragments withsarkosyl. In the presence of this detergent, lipid-modified proteinsremain soluble while the outer membrane fragments are precipitated andcan be removed by ultracentrifugation.

[0143] Labelling of Proteins with [³H]Palmitate and GlobomycinTreatment.

[0144] Exponentially growing cells (4×10⁸ cells per ml) of H. somnusstrain HS25 in BHI-TT and of E. coli DH5αF′IQ harboring the specifiedplasmids in M63 defined medium were incubated for 2 h at 37° C. with[H]palmitate at a final concentration of 50 μCi/ml, in the absence orpresence of globomycin (100 μg/ml), a specific inhibitor ofprolipoprotein signal peptidase II (Dev et al. (1985) J. Biol. Chem.260:5891-5894) as described previously (Theisen et al. (1992) Infect.Immun. 62:826-831). Labelling was terminated by precipitation withtrichloroacetic acid (10%, wt/vol) for 30 min on ice. Proteins werepelleted by centrifugation at 15,000× g for 20 min and washed twice withmethanol to remove lipids. The proteins, resuspended in sample buffer,were analyzed by SDS-PAGE and the radiolabelled protein bands in thedried gel were detected by fluorography.

[0145] Fractionation of H. somnus Cells and Preparation of OuterMembranes.

[0146] Exponentionally growing H. somnus HS25 cells were lysed by twopassages through a French pressure cell. Separation of the variouscellular fractions, including Sarkosyl-insoluble outer membranes (Filipet al. (1973) J. Bacteriol. 115:717-722) was done by differentialcentrifugation as previously described (Rioux et al. (1992) Gene116:13-20). The proteins from cell lysates and various fractions wereprecipitated at 10% (wt/vol) trichloroacetic acid for 40 min on ice,pelleted by centrifugation at 15,000× g for 20 min, and washed twicewith methanol to remove lipids before analysis by SDS-PAGE.

Results

[0147] In order to identify clones expressing Tbp epitopes, a genomicexpression library of H. somnus strain HS25 in E. coli was screened withpolyclonal antiserum raised against affinity-purified Tbp1 and Tbp2 ofH. somnus. This anti-Tbp antiserum reacted with proteins with relativemolecular weights of 80,000 and 115,000, respectively (termed Tbp2 andTbp1, respectively, herein).

[0148] A clone carrying a 4.1-kilobase pair DNA insert was obtained. Theanalysis of the nucleotide sequence of the DNA insert showed thepresence of a truncated open reading frame coding for a predictedpolypeptide similar to the carboxyl region of predicted Tbp1polypeptides of Neisseria meningitidis and Neisseria gonorrhoeae. Apolypeptide with M_(r) of approximately 110,000 was produced by theclone; this polypeptide was recognized by bovine hyperimmune serumagainst live H. somnus HS25.

[0149] The DNA region coding for the amino terminus of H. somnus Tbp1was obtained by using the method of inverse polymerase chain reaction.The complete tbp1 ORF codes for a 971 amino acid polypeptide withpredicted molecular weight of 109,725. The reading frame and a putativecleavage site of signal peptidase I were confirmed by the partial aminoacid sequence obtained from N-terminal microsequencing of the matureform of native H. somnus Tbp1. The molecule includes a signal peptide of28 amino acids.

[0150] The tbp1 gene region coding for the mature Tbp1 was subclonedinto an E. coli expression vector pGH432, containing a tac promoter togive plasmid pCRR41 (ATCC Accession No. 98810) which expressed the H.somnus Tbp1 protein as insoluble inclusion bodies following inductionwith IPTG, and Tbp1 was partially purified by aggregate preparation.

[0151] The gene coding for Tbp2 was isolated by inverse PCR and thesequence coding for the entire Tbp2 peptide, including the signalsequence, was expressed in the same vector as described above. Thisplasmid was named pCRR90 (ATCC Accession No. 98811). Following IPTGinduction, the Tbp2 protein was extracted from total E. coli outermembranes with Sarkosyl, as described above. Unlike other membraneproteins, Tbp2 remained soluble in this detergent due to its lipidmodification.

[0152] The genes coding for Tbp1 and Tbp2, plus flanking DNA are shownin FIGS. 1A-1B. Two open reading frames were found, one starting atnucleotide 708 and ending at position 2693 (Tbp2) and the secondstarting at nucleotide 2891 and ending at position 5803 (Tbp1) (see FIG.2). The predicted amino acid sequences of these two proteins are shownin FIG. 3 (Tbp1) and FIG. 4 (Tbp2). The full-length Tbp1 sequenceincludes a signal peptide of 28 amino acids, occurring at positions 1 to28 of FIG. 3. Thus, the mature Tbp1 sequence is represented by aminoacids 29 to 971, inclusive, of FIG. 3 and is encoded by the nucleotidesequence depicted at positions 2975 to 5803, inclusive of FIGS. 1A-1B.

[0153] The full-length Tbp2 sequence includes a signal peptide of 19amino acids, occurring at positions 1 to 19 of FIG. 4. Thus, the matureTbp2 sequence is represented by amino acids 20 to 662, inclusive, ofFIG. 4 and is encoded by the nucleotide sequence depicted at positions765 to 2683, of FIGS. 1A-1B.

EXAMPLE 2 Protective Efficacy of Recombinant Transferrin-BindingProteins

[0154] The Tbp1 and Tbp2 proteins were produced recombinantly in E. colias inclusion bodies and as a membrane bound protein, respectively. Asexplained above, Tbp1 inclusion bodies were prepared using standardprocedures while soluble Tbp2 was prepared from E. coli outer membranes.These membranes were then subjected to a sarkosyl extraction in order topreferentially solubilize Tbp2.

[0155] Vaccines were formulated using the adjuvant VSA3 (a combinationof DDA (Kodak) and Emulsigen-Plus (MVP Laboratories, Omaha, Nebr.)) suchthat the volume of each dose was 2 cc containing 50 μg of each antigen.A placebo vaccine was also prepared containing sterile diluent in placeof antigen. Three groups were included in the trial, one of whichreceived placebo, a second which received two immunizations with Tbp2and a third which received two immunizations with Tbp1+Tbp2. Each grouphad eight animals and the interval between primary and secondaryimmunization was three weeks. All vaccinations were carried out at afarm in southern Saskatchewan and vaccines were delivered via thesubcutaneous route.

[0156] Two weeks after the second immunization, animals were challengedwith bovine herpesvirus-1 followed four days later by aerosol exposureto H. somnus strain HS25. Animals were examined daily by a veterinarianand animal health technician and the following data was recorded:weight, temperature, nasal scores, depression, strength, respiratorydistress and sickness. Each of these criteria, with the exception ofweight and temperature, was scored on a scale of 0-4.

[0157] The serological response to vaccination was measured using anenzyme-linked immunosorbent assay (ELISA). Serum samples were collectedat the time of the first and second immunizations plus on the day ofchallenge with BHV-1. The titers are presented as the reciprocal of theserum dilution which resulted in an optical density equivalent to thebackground plus two standard deviations.

[0158] None of the animals showed any adverse response to immunizationwith any of the formulations used. The serological response tovaccination was determined using an ELISA procedure which measured theserum antibody levels to Tbp1 and Tbp2. An H. somnus outer membraneextract was also used as an antigen but no significant increase in titerwas observed. This is not unexpected, since the level of iron-regulatedouter membrane proteins in this antigen preparation is extremely low.

[0159] The antibody titers against Tbp1 and Tbp2 are shown in FIGS. 5and 6, respectively. It can be seen that animals receiving recombinantTbp2 vaccines responded well to this antigen, with no significantdifference between Groups 2 and 3. The response against Tbp1 wasminimal, as expected based on our experience with this antigen from ourother organisms. The group which received only Tbp2 also had serumantibody levels against Tbp1, but this was probably due to contaminatingE. coli proteins present in the antigen preparation used for the ELISA.

[0160] Mortality in the placebo group was 62.5%, close to an expectedrate of approximately 70%. The mortality by group is shown in FIG. 7 andis listed by day in Table 1. As can be seen, immunization with vaccinescontaining recombinant Tbp2 reduced mortality to 25% while immunizationwith vaccines including a combination of Tbp1 and Tbp2, had littleeffect compared to the placebo. Necropsies were performed on all animalswhich died during the trial and in all cases, H. somnus was culturedfrom the lungs and the pathology observed was consistent with H. somnuspneumonia.

[0161] Since the ELISA titers to Tbp2 were similar in both of theexperimental vaccine groups, it is surprising that equivalent levels ofprotection were not observed. However, this may simply reflect moreefficient uptake of H. somnus by phagocytic cells in the Tbp1+Tbp2group, allowing for increased multiplication of the bacteria in anintracellular environment.

[0162] The clinical results are summarized in Table 1 and the resultsfor temperature, depression, and sick scores are illustrated in FIGS. 8,9 and 10, respectively. These results are similar to those obtained formortality, with the Tbp2-immunized group showing consistently lowerscores in virtually all categories. The results shown in FIGS. 8, 9 and10 only include days 5 through 8 of the trial since animals werechallenged with H. somnus on day 4. The clinical scores were virtuallyidentical between all three groups on days 1 through 4. TABLE 1 Meanclinical scores and mortality by group. Group Day Weight Temp. NasalDep. Str. Resp. Sick Cumulative Mortality Placebo 0 259 38.96 0.00 0.000.00 0.00 0.00 0 Placebo 1 258 39.13 0.00 0.00 0.00 0.00 0.00 0 Placebo2 250 40.94 0.88 0.38 0.00 0.00 1.00 0 Placebo 3 248 41.64 1.25 0.630.00 0.00 1.38 0 Placebo 4 244 40.53 1.75 0.88 0.00 0.38 1.25 0 Placebo5 239 40.64 2.38 1.25 1.00 0.88 1.75 0 Placebo 6 237 40.77 2.88 1.881.88 2.00 2.75 3 Placebo 7 251 40.15 2.50 1.50 1.25 1.25 2.00 5 Placebo8 259 39.57 1.33 0.67 0.33 0.33 0.67 5 Tbp2 0 274 39.01 0.06 0.00 0.000.00 0.00 0 Tbp2 1 268 39.19 0.13 0.00 0.00 0.00 0.00 0 Tbp2 2 262 41.051.25 0.13 0.00 0.00 1.00 0 Tbp2 3 257 41.06 1.13 0.38 0.00 0.00 1.00 0Tbp2 4 253 40.68 1.63 0.75 0.00 0.75 1.25 0 Tbp2 5 250 40.10 1.25 1.000.63 0.50 1.25 1 Tbp2 6 246 40.26 2.00 1.43 1.00 1.29 1.57 1 Tbp2 7 25439.63 1.33 0.67 0.33 0.50 0.83 2 Tbp2 8 253 39.42 0.50 0.33 0.33 0.170.33 2 Tbp1 + Tbp2 0 259 38.95 0.00 0.00 0.00 0.00 0.00 0 Tbp1 + Tbp2 1251 39.16 0.00 0.00 0.00 0.00 0.00 0 Tbp1 + Tbp2 2 244 40.70 0.75 0.130.00 0.00 0.88 0 Tbp1 + Tbp2 3 241 41.39 1.25 0.38 0.00 0.00 1.13 0Tbp1 + Tbp2 4 241 40.43 1.50 0.75 0.00 0.38 1.25 0 Tbp1 + Tbp2 5 23540.09 1.63 0.75 0.38 0.38 1.13 0 Tbp1 + Tbp2 6 236 40.47 2.00 1.14 0.711.00 1.43 1 Tbp1 + Tbp2 7 235 40.24 2.00 1.57 1.14 1.57 2.00 3 Tbp1 +Tbp2 8 249 39.68 0.75 0.00 0.00 0.00 0.50 4

[0163] Deposits of Strains Useful in Practicing the Invention

[0164] A deposit of biologically pure cultures of the following strainswas made with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas. The accession number indicated was assigned aftersuccessful viability testing, and the requisite fees were paid. Thedeposits were made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for a period ofthirty (30) years from the date of deposit. The organisms will be madeavailable by the ATCC under the terms of the Budapest Treaty, whichassures permanent and unrestricted availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 U.S.C. §122 and the Commissioner'srules pursuant thereto (including 37 C.F.R. §1.12 with particularreference to 886 OG 638). Upon the granting of a patent, allrestrictions on the availability to the public of the deposited cultureswill be irrevocably removed.

[0165] These deposits are provided merely as convenience to those ofskill in the art, and are not an admission that a deposit is requiredunder 35 U.S.C. §112. The nucleic acid sequences of these genes, as wellas the amino acid sequences of the molecules encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with the description herein. Strain Deposit Date ATCC No.pCRR41 in E. coli DH5alphaF′IQ Jul. 14, 1998 98810 pCRR90 in E. coliDH5alphaF′IQ Jul. 14, 1998 98811

[0166] Thus, the cloning, expression and characterization of H. somnustransferrin-binding proteins are disclosed, as are methods of using thesame. Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1 5 1 6376 DNA Haemophilus somnus 1 aagcttgcat aattgcttca acgccttatcactataccgt aaagtgtaca tcactcaatt 60 cctaacatct tgcatacctc tgcgtgagaataactcattg gggttttgtt gtagtcttcc 120 aaagactcac gatatactgc aaggtcatattcatcttcga ttttttcaaa aacggcattc 180 ctgaacagtt cggatagcgt aatattatttgtttttgcat aggacttaaa taattgctcg 240 tcttgagcgt ttagtcttac tgaaatagccatagtaaaat ttcctttcat tttgtattac 300 attgtaatac atttatacag aatttgcaatataggtgaaa aaagaaacag agattagacg 360 tggtcgacac gttgttttta actgacacgttcatttggtt ttcgtgacaa aatatcgcag 420 agaagttttt acggcacgat tagataaaaaattaaattta aagagaaaat ctcgcaaggt 480 aaaaaccgtc agctaacgtt gttggaaatgattgcccgcc ctcagatcga ccaaaatcgc 540 acgtcttaat cgttccgagt gctgtatcgttacaacaaac agcggctgtt taaggaatcc 600 atcagcagcg tgggcgtgtt gaaaactgtatgcattcctt aaaaaaatac atataataat 660 aatttttatt tgcatatttt atataatataagaaaggata taggtaaatg acctctttca 720 aattattagg cttttcggtc ttgagtgtggctttgctctc tgcctgctct tccggcaaag 780 gtggctttga tttagacgac gtcgagcatacccccccctc ctcctcgggt agttcccgcc 840 ccacttatca agatgttccg actggacaacggcagcaaga aatagtagaa gaaatcaact 900 cacctgctct aggttatgcg acagaaattccgcgtaggaa tatttcgcca atgcccacca 960 cgggcacaaa agaaagtaat gctcgtgttgccattactgc ccagcaagtt gcccctctta 1020 gcatgccttt taattcaata aaagaagattttatcaaaag gctaatagca gaaaacacca 1080 agaaaaatgc acgaggtaga gatgtaaaatattttgatga tacagatgac gtgttgtttg 1140 cacatgatgg atctaattta gcgcataaacgtgatttaca atatgttcga gttgggtatg 1200 tgttgggtac ccgaaagatt gaacttgttttttcccatga taaaaaaaca agagatcaat 1260 ttcctgctgg ttgggtaggt tatgttttttaccaaggcac tagccctgcg gttacattac 1320 ctacacaaac cgtaacatac aaaggctattgggattttgt tagtgatgcc ttcaatgaac 1380 gaactttagc tgaagatttt acacaggaaaatagttctgc tactagcaat ataccgggca 1440 atcaaatcgg tgcgacttca atggatgcattggttaatcg caaagtttca ggagaaaaaa 1500 tcaatattgc tcacagtgct gagtttactgctgattttgg cagtaaaaaa ctttcaggtg 1560 aattaaaaag taacggttat gtttctagaatagaaaatga gcaacaagat gtaaaaacac 1620 gctacaaaat tgatgctgat atcaaaggcaaccgttttgt cggttcagca acagcacaag 1680 aaaaaagtca caccatcttc ggcaaggatgcggacaaacg tctcgagggc ggtttctttg 1740 gtcctaaagc cgaggaactg gcaggtaaatttctgaccga tgataattcc ctgtttgtcg 1800 tctttggtgc aaaacgtgaa agtaaaggcgatgaaaaact agaaacccgc tttgatgccg 1860 ttaaaatcag cacggatagt aataaattagaaaaagaaac gatggatacg ttcggcaatg 1920 cggcgtattt agtgttggac ggcagacagtttcctttggt gccggaaagt aatgccggta 1980 cgacgggcgc tggcaacaca ggcaagaatgagtttatcag caccatagac ggtagccatt 2040 taaacaaaac aaatcatgaa aaccacaaaaaatacaaagt taccgtatgt tgcagtaatt 2100 tggtgtatgt gaaattcggc agctatggggaacaaacaac agcaaacgat gcttcaaaca 2160 gcactgccgg tgcagcaact acgcataacagcacattaac aaacggacac cttttcctaa 2220 caggtgaacg tacttcgctt actgatatggcgaagcaaag tggtgcagca aagtatattg 2280 gcacatggca agccaacttt ttaagtagcaaaggacaggt tggcagtgtt gacgccggtg 2340 atccgcgtaa cgatagtggt aaaagccgtgctgaatttga tgttaatttt ggtggtaaga 2400 cagttacagg caagtttttt gatgccgacggtattcaacc cgccctcaca atggatagta 2460 ccaagattga aggtaacggc ttctcgggtacagctaagac aactggtagt ttgcaattag 2520 ataaaggcag tacaggtgcg ggtataacagtaaccttcac cgatgctaaa gtcgatggcg 2580 cattctacgg tccgaatgca gaagaaatcggcggtaccat cacatcgaat ggcacgggcg 2640 ataaagtcgg tggggtgttc ggtgcgaaacgccaagaact atcgcaacag aaatgaaatc 2700 ttaactctag ctacctgaaa catatttcaggtagcaggat gggtatctgc tgatatgctc 2760 aacctgcttg aaaaagtttg tcaaatccgccgcctgtcgt tgttgacggt gtgatgttgc 2820 agtggcaaat cgctcggctt tgttgagaaagcatgaccca tccgtctttt actcacacgg 2880 aacgaaaaaa atgtctacaa aacctttgtttaaacttaag ctgataacat tggctgtcag 2940 cacgattttt ttacctttta ctgaggcggttgccgatact gaatcaccga gtagcaatac 3000 agaagcagtg ctggagttag aagctatccaggtgcaagcc aaacacgaga tcagcagaca 3060 tgacaatgaa gtcaccggtt tgggtaaggtggtcaaaagc agtgaagaca ttgataaaga 3120 actgattttg aatattcgcg atttgacccgttatgatccc ggtatttcgg tggtggagca 3180 gggacgtggt gcaacgtcag gctatgcaatgcgtggtgtt gacagaaacc gcgtggctat 3240 gttggtggac ggcttgggac aggcgcagtcctattctacc ttgaaatccg atgccaacgg 3300 cggggcgatt aatgaaattg aatatgagaatattaaatca attgaattga gcaaggggtc 3360 cagttcggca gaatacggta gcggtgccttgggcggtgcg gtagggtttc gtaccaaaga 3420 agctgatgat gtgattaaag aggggcaaaactggggcttg aacagtaaaa cggcttacag 3480 cagcaaaaac agccagttta cccaatccgttgccggtgcg ttccgtgtcg gcggttttga 3540 cagtttggcg atttttaccc atcgtaaaggtaaggaaacc cgcgtgcatc ctgctgccga 3600 agaaatacaa catacttacc aaccattggaagggtatttt aatcggtatg aggttgacca 3660 aaaccgcaac ggaaagcctg ttctggcgaatgcgtattat atacttgccg atgaatgctc 3720 taatctaagt gatccgagtt gtcgtcatgccaaggccaag acgaataggg tgggtgcccc 3780 ggagaacaat cctaattgga cgcccgaagagcaggcacag gctgctaaaa tgccgtatcc 3840 gacacgtacc gcctctgcca aagattatacgggtcctgac cgcatcagcc ctaatccgat 3900 ggactaccaa agtcactctt tcttctggaaaggtggttac cgcttgtcgc ctaaccatta 3960 tgtcggcggg gtgttggaac atacgaagcagcgttacgat atccgtgata tgacgcaacg 4020 ggcgtattac acgaaagagg atatctgccacagcggatcc agttgccaaa cgttggataa 4080 aaatgagacg gacaaaggta atttcggtatcacgttgact gataatcctt tggacggttt 4140 ggtatatgat gccggcaatc aagctcgtggcgtgcggtac ggacggggta aattttttaa 4200 tgaacgccat acgaaaaatc gctcgggtatcttttaccgc tatgagaatc ccgataaaaa 4260 ttcttggcca gatagcttga ccttgagtattgaccgccaa gatctcaaac tgtcgagccg 4320 tatccattgg acgtattgca ccgattatcctcatgtggca cgttgccgtg ccagcttgga 4380 caaaccttgg tctaattacc gtaccgagaaaaacgattat caagaacgac tcaatctggg 4440 acaattcaat tgggaaaaaa cttttaatctgggctttacc acgcataagg tgaatatcgc 4500 cgccggcttt ggtacacatc gctccaccttacaacatggc gacttatatg ctgaatatgt 4560 caccttgcca ccgtatacag aggaaaaagtgtatggcgaa gataataagg tcaaacaaaa 4620 tccgacagca gaagaaaaag agaaattacaatacggcaat ggttcttatg acaaacctcg 4680 cgtatataga cgtaaaaaca cgccggaattaaaaactgtc aatgggtgca atgagacagc 4740 aggcgataac cgtgactgct cgccacgtgtgattacgggc agacagtatt accttgcctt 4800 gcgtaaccat attgcctttg gtgaatgggcagacttgggg ttgggcgtgc ggtacgacaa 4860 ccataccttc cgctcgaatg acccgtggaccaaaggtggc aactaccaca actggtcgtg 4920 gaatgcgggc gtgagcctca aaccaacccgccactttgtc gtgtcttacc gtgtgtccag 4980 cggtttccgt gtccccgctt tttatgagctgtacggcgtg cgtacggggg cttctggtaa 5040 agacaatcca ctcacacaaa aagagttcttgagccgtaaa ccgttgaaaa gcgaaaaagc 5100 ctttaaccaa gaaattggtt tggccgttcagggcgatttt ggtgtgatag agaccagttt 5160 cttccaaaac aactataaaa acctgcttgcccgtgcagat aaatatgtcg agggattggg 5220 ttatgtaacc gatttttaca acacccaagatgtcaaactc aacggtatca atatcttggg 5280 tagaatctac tgggaaggca tcagcgataggctgcctgaa ggcttgtatt ccacacttgc 5340 ttacaaccgt atcaatatca aagcacgcaaattgcacgac aattttacca atgtgtctga 5400 gccgacattg gaagcagtgc aaccgggacgcattattgca agtatcggct atgatgaccc 5460 tgagggcaga tggggcctta atttaagcggcacctactct caagccaaac aacgtgacga 5520 agtggtcggc gaaaaagtgt tcggcaagggtggcagcatt aaacggacga tcaacagcaa 5580 acgcactcgt gcttggtata tttatgatttgacggcatac tacacttgga aagaaaaatt 5640 cacgttgaga gccggtatct ataatttaaccaatcgtaaa tatagcacat gggaaagtgt 5700 gcgtcagtcc gctgccaatg cggtcaatcaagacctaggt acacgttcgg cacgttttgc 5760 cgcacggggc agaaacttta ccgtgagtatggaaatgaag ttttaattaa aaaactgtct 5820 gcaagctgtt taaaaaacag ttaagatgatttgttcgtat aaatagctgc ctgaagtctt 5880 gttatgcagg ttcaggcagc ttgacattacaaaaaaagga aaaaagtcta atggaagaca 5940 aatatgctat ttgtcggcaa cgaaaaattgttgcaatgga ttagcagttc aagtggcttc 6000 cggtattttt tatcgcactt tttctatctataagcttgaa atctttattt ccgaacttat 6060 attttcgctg tttgttaatt tcactattggaaaaggaaat attatgtcaa caaatcaaga 6120 aacacgtggt tttcagtctg aagttaaacagcttttacaa ttgatgattc attctcttta 6180 ttcaaataaa gagatttttt tgcgtgagttgatttccaat gcgtctgatg cggcggataa 6240 attgcgtttt aaagccttgt ctgcacctgaattatatgaa ggagatggtg atttaaaagt 6300 gcggatcagt tttgacgcag agaaaggtacgttaaccatt agcgataatg gtattggtat 6360 gacgagagag caggtg 6376 2 971 PRTHaemophilus somnus 2 Met Ser Thr Lys Pro Leu Phe Lys Leu Lys Leu Ile ThrLeu Ala Val 1 5 10 15 Ser Thr Ile Phe Leu Pro Phe Thr Glu Ala Val AlaAsp Thr Glu Ser 20 25 30 Pro Ser Ser Asn Thr Glu Ala Val Leu Glu Leu GluAla Ile Gln Val 35 40 45 Gln Ala Lys His Glu Ile Ser Arg His Asp Asn GluVal Thr Gly Leu 50 55 60 Gly Lys Val Val Lys Ser Ser Glu Asp Ile Asp LysGlu Leu Ile Leu 65 70 75 80 Asn Ile Arg Asp Leu Thr Arg Tyr Asp Pro GlyIle Ser Val Val Glu 85 90 95 Gln Gly Arg Gly Ala Thr Ser Gly Tyr Ala MetArg Gly Val Asp Arg 100 105 110 Asn Arg Val Ala Met Leu Val Asp Gly LeuGly Gln Ala Gln Ser Tyr 115 120 125 Ser Thr Leu Lys Ser Asp Ala Asn GlyGly Ala Ile Asn Glu Ile Glu 130 135 140 Tyr Glu Asn Ile Lys Ser Ile GluLeu Ser Lys Gly Ser Ser Ser Ala 145 150 155 160 Glu Tyr Gly Ser Gly AlaLeu Gly Gly Ala Val Gly Phe Arg Thr Lys 165 170 175 Glu Ala Asp Asp ValIle Lys Glu Gly Gln Asn Trp Gly Leu Asn Ser 180 185 190 Lys Thr Ala TyrSer Ser Lys Asn Ser Gln Phe Thr Gln Ser Val Ala 195 200 205 Gly Ala PheArg Val Gly Gly Phe Asp Ser Leu Ala Ile Phe Thr His 210 215 220 Arg LysGly Lys Glu Thr Arg Val His Pro Ala Ala Glu Glu Ile Gln 225 230 235 240His Thr Tyr Gln Pro Leu Glu Gly Tyr Phe Asn Arg Tyr Glu Val Asp 245 250255 Gln Asn Arg Asn Gly Lys Pro Val Leu Ala Asn Ala Tyr Tyr Ile Leu 260265 270 Ala Asp Glu Cys Ser Asn Leu Ser Asp Pro Ser Cys Arg His Ala Lys275 280 285 Ala Lys Thr Asn Arg Val Gly Ala Pro Glu Asn Asn Pro Asn TrpThr 290 295 300 Pro Glu Glu Gln Ala Gln Ala Ala Lys Met Pro Tyr Pro ThrArg Thr 305 310 315 320 Ala Ser Ala Lys Asp Tyr Thr Gly Pro Asp Arg IleSer Pro Asn Pro 325 330 335 Met Asp Tyr Gln Ser His Ser Phe Phe Trp LysGly Gly Tyr Arg Leu 340 345 350 Ser Pro Asn His Tyr Val Gly Gly Val LeuGlu His Thr Lys Gln Arg 355 360 365 Tyr Asp Ile Arg Asp Met Thr Gln ArgAla Tyr Tyr Thr Lys Glu Asp 370 375 380 Ile Cys His Ser Gly Ser Ser CysGln Thr Leu Asp Lys Asn Glu Thr 385 390 395 400 Asp Lys Gly Asn Phe GlyIle Thr Leu Thr Asp Asn Pro Leu Asp Gly 405 410 415 Leu Val Tyr Asp AlaGly Asn Gln Ala Arg Gly Val Arg Tyr Gly Arg 420 425 430 Gly Lys Phe PheAsn Glu Arg His Thr Lys Asn Arg Ser Gly Ile Phe 435 440 445 Tyr Arg TyrGlu Asn Pro Asp Lys Asn Ser Trp Pro Asp Ser Leu Thr 450 455 460 Leu SerIle Asp Arg Gln Asp Leu Lys Leu Ser Ser Arg Ile His Trp 465 470 475 480Thr Tyr Cys Thr Asp Tyr Pro His Val Ala Arg Cys Arg Ala Ser Leu 485 490495 Asp Lys Pro Trp Ser Asn Tyr Arg Thr Glu Lys Asn Asp Tyr Gln Glu 500505 510 Arg Leu Asn Leu Gly Gln Phe Asn Trp Glu Lys Thr Phe Asn Leu Gly515 520 525 Phe Thr Thr His Lys Val Asn Ile Ala Ala Gly Phe Gly Thr HisArg 530 535 540 Ser Thr Leu Gln His Gly Asp Leu Tyr Ala Glu Tyr Val ThrLeu Pro 545 550 555 560 Pro Tyr Thr Glu Glu Lys Val Tyr Gly Glu Asp AsnLys Val Lys Gln 565 570 575 Asn Pro Thr Ala Glu Glu Lys Glu Lys Leu GlnTyr Gly Asn Gly Ser 580 585 590 Tyr Asp Lys Pro Arg Val Tyr Arg Arg LysAsn Thr Pro Glu Leu Lys 595 600 605 Thr Val Asn Gly Cys Asn Glu Thr AlaGly Asp Asn Arg Asp Cys Ser 610 615 620 Pro Arg Val Ile Thr Gly Arg GlnTyr Tyr Leu Ala Leu Arg Asn His 625 630 635 640 Ile Ala Phe Gly Glu TrpAla Asp Leu Gly Leu Gly Val Arg Tyr Asp 645 650 655 Asn His Thr Phe ArgSer Asn Asp Pro Trp Thr Lys Gly Gly Asn Tyr 660 665 670 His Asn Trp SerTrp Asn Ala Gly Val Ser Leu Lys Pro Thr Arg His 675 680 685 Phe Val ValSer Tyr Arg Val Ser Ser Gly Phe Arg Val Pro Ala Phe 690 695 700 Tyr GluLeu Tyr Gly Val Arg Thr Gly Ala Ser Gly Lys Asp Asn Pro 705 710 715 720Leu Thr Gln Lys Glu Phe Leu Ser Arg Lys Pro Leu Lys Ser Glu Lys 725 730735 Ala Phe Asn Gln Glu Ile Gly Leu Ala Val Gln Gly Asp Phe Gly Val 740745 750 Ile Glu Thr Ser Phe Phe Gln Asn Asn Tyr Lys Asn Leu Leu Ala Arg755 760 765 Ala Asp Lys Tyr Val Glu Gly Leu Gly Tyr Val Thr Asp Phe TyrAsn 770 775 780 Thr Gln Asp Val Lys Leu Asn Gly Ile Asn Ile Leu Gly ArgIle Tyr 785 790 795 800 Trp Glu Gly Ile Ser Asp Arg Leu Pro Glu Gly LeuTyr Ser Thr Leu 805 810 815 Ala Tyr Asn Arg Ile Asn Ile Lys Ala Arg LysLeu His Asp Asn Phe 820 825 830 Thr Asn Val Ser Glu Pro Thr Leu Glu AlaVal Gln Pro Gly Arg Ile 835 840 845 Ile Ala Ser Ile Gly Tyr Asp Asp ProGlu Gly Arg Trp Gly Leu Asn 850 855 860 Leu Ser Gly Thr Tyr Ser Gln AlaLys Gln Arg Asp Glu Val Val Gly 865 870 875 880 Glu Lys Val Phe Gly LysGly Gly Ser Ile Lys Arg Thr Ile Asn Ser 885 890 895 Lys Arg Thr Arg AlaTrp Tyr Ile Tyr Asp Leu Thr Ala Tyr Tyr Thr 900 905 910 Trp Lys Glu LysPhe Thr Leu Arg Ala Gly Ile Tyr Asn Leu Thr Asn 915 920 925 Arg Lys TyrSer Thr Trp Glu Ser Val Arg Gln Ser Ala Ala Asn Ala 930 935 940 Val AsnGln Asp Leu Gly Thr Arg Ser Ala Arg Phe Ala Ala Arg Gly 945 950 955 960Arg Asn Phe Thr Val Ser Met Glu Met Lys Phe 965 970 3 662 PRTHaemophilus somnus 3 Met Thr Ser Phe Lys Leu Leu Gly Phe Ser Val Leu SerVal Ala Leu 1 5 10 15 Leu Ser Ala Cys Ser Ser Gly Lys Gly Gly Phe AspLeu Asp Asp Val 20 25 30 Glu His Thr Pro Pro Ser Ser Ser Gly Ser Ser ArgPro Thr Tyr Gln 35 40 45 Asp Val Pro Thr Gly Gln Arg Gln Gln Glu Ile ValGlu Glu Ile Asn 50 55 60 Ser Pro Ala Leu Gly Tyr Ala Thr Glu Ile Pro ArgArg Asn Ile Ser 65 70 75 80 Pro Met Pro Thr Thr Gly Thr Lys Glu Ser AsnAla Arg Val Ala Ile 85 90 95 Thr Ala Gln Gln Val Ala Pro Leu Ser Met ProPhe Asn Ser Ile Lys 100 105 110 Glu Asp Phe Ile Lys Arg Leu Ile Ala GluAsn Thr Lys Lys Asn Ala 115 120 125 Arg Gly Arg Asp Val Lys Tyr Phe AspAsp Thr Asp Asp Val Leu Phe 130 135 140 Ala His Asp Gly Ser Asn Leu AlaHis Lys Arg Asp Leu Gln Tyr Val 145 150 155 160 Arg Val Gly Tyr Val LeuGly Thr Arg Lys Ile Glu Leu Val Phe Ser 165 170 175 His Asp Lys Lys ThrArg Asp Gln Phe Pro Ala Gly Trp Val Gly Tyr 180 185 190 Val Phe Tyr GlnGly Thr Ser Pro Ala Val Thr Leu Pro Thr Gln Thr 195 200 205 Val Thr TyrLys Gly Tyr Trp Asp Phe Val Ser Asp Ala Phe Asn Glu 210 215 220 Arg ThrLeu Ala Glu Asp Phe Thr Gln Glu Asn Ser Ser Ala Thr Ser 225 230 235 240Asn Ile Pro Gly Asn Gln Ile Gly Ala Thr Ser Met Asp Ala Leu Val 245 250255 Asn Arg Lys Val Ser Gly Glu Lys Ile Asn Ile Ala His Ser Ala Glu 260265 270 Phe Thr Ala Asp Phe Gly Ser Lys Lys Leu Ser Gly Glu Leu Lys Ser275 280 285 Asn Gly Tyr Val Ser Arg Ile Glu Asn Glu Gln Gln Asp Val LysThr 290 295 300 Arg Tyr Lys Ile Asp Ala Asp Ile Lys Gly Asn Arg Phe ValGly Ser 305 310 315 320 Ala Thr Ala Gln Glu Lys Ser His Thr Ile Phe GlyLys Asp Ala Asp 325 330 335 Lys Arg Leu Glu Gly Gly Phe Phe Gly Pro LysAla Glu Glu Leu Ala 340 345 350 Gly Lys Phe Leu Thr Asp Asp Asn Ser LeuPhe Val Val Phe Gly Ala 355 360 365 Lys Arg Glu Ser Lys Gly Asp Glu LysLeu Glu Thr Arg Phe Asp Ala 370 375 380 Val Lys Ile Ser Thr Asp Ser AsnLys Leu Glu Lys Glu Thr Met Asp 385 390 395 400 Thr Phe Gly Asn Ala AlaTyr Leu Val Leu Asp Gly Arg Gln Phe Pro 405 410 415 Leu Val Pro Glu SerAsn Ala Gly Thr Thr Gly Ala Gly Asn Thr Gly 420 425 430 Lys Asn Glu PheIle Ser Thr Ile Asp Gly Ser His Leu Asn Lys Thr 435 440 445 Asn His GluAsn His Lys Lys Tyr Lys Val Thr Val Cys Cys Ser Asn 450 455 460 Leu ValTyr Val Lys Phe Gly Ser Tyr Gly Glu Gln Thr Thr Ala Asn 465 470 475 480Asp Ala Ser Asn Ser Thr Ala Gly Ala Ala Thr Thr His Asn Ser Thr 485 490495 Leu Thr Asn Gly His Leu Phe Leu Thr Gly Glu Arg Thr Ser Leu Thr 500505 510 Asp Met Ala Lys Gln Ser Gly Ala Ala Lys Tyr Ile Gly Thr Trp Gln515 520 525 Ala Asn Phe Leu Ser Ser Lys Gly Gln Val Gly Ser Val Asp AlaGly 530 535 540 Asp Pro Arg Asn Asp Ser Gly Lys Ser Arg Ala Glu Phe AspVal Asn 545 550 555 560 Phe Gly Gly Lys Thr Val Thr Gly Lys Phe Phe AspAla Asp Gly Ile 565 570 575 Gln Pro Ala Leu Thr Met Asp Ser Thr Lys IleGlu Gly Asn Gly Phe 580 585 590 Ser Gly Thr Ala Lys Thr Thr Gly Ser LeuGln Leu Asp Lys Gly Ser 595 600 605 Thr Gly Ala Gly Ile Thr Val Thr PheThr Asp Ala Lys Val Asp Gly 610 615 620 Ala Phe Tyr Gly Pro Asn Ala GluGlu Ile Gly Gly Thr Ile Thr Ser 625 630 635 640 Asn Gly Thr Gly Asp LysVal Gly Gly Val Phe Gly Ala Lys Arg Gln 645 650 655 Glu Leu Ser Gln GlnLys 660 4 2179 DNA Haemophilus somnus CDS (872)..(1906) 4 cgacgccagtgccaagcttg catgcctgca ggtgatctaa gcttcccggg atccaagagg 60 tgaagagatttattggattg gaccaatagg actggcagaa aatgaatcgg aaggaacgga 120 cttccatgccgttaaaaacg gctatgtgtc aattacaccc attcaaacag atatgacggc 180 atatcattcaatgacagctt tacaacaatg gttagataag gaataacgat aatcttttca 240 tcgaaggaataaaacatgaa aattttcggt acgctatatg ataaaactat gcaatgggca 300 aatcaccgttttgctacatt ttggctaact tttgttagtt ttattgaggc tattttcttc 360 ccaataccacctgatgtcat gcttattccg atgtcaataa ataaacctaa atgtgctact 420 aaatttgcattttatgcagc aatggcttca gccattggtg gggcaattgg ttatggatta 480 ggttattacgcttttgattt catacaaagt tatattcaac aatggggtta tcaacaacat 540 tgggaaactgctctttcttg gttcaaagaa tggggtattt gggtagtttt cgttgcaggt 600 ttttcacctattccttataa aatttttacg atttgtgcag gcgtcatgca aatggcattt 660 ttgcctttcttacttactgc ctttatttct cgtattgcaa gatttttgct cgttacccat 720 ttagcggcttggagcggaaa aaaatttgct gcgaaattac gtcaatctat tgaatttatc 780 ggttggtcagttgtcattat tgctatagtt gtatatcttg tcttgaaata atctaagata 840 aaaaatgaatataaagtaac ggagaattta c atg aaa aaa ttt tta cct tta 892 Met Lys Lys PheLeu Pro Leu 1 5 tct att agt atc act gta cta gct gct tgt agt tca cac actccg gct 940 Ser Ile Ser Ile Thr Val Leu Ala Ala Cys Ser Ser His Thr ProAla 10 15 20 ccg gta gaa aat gct aag gat tta gca cca agt att atc aaa ccgatt 988 Pro Val Glu Asn Ala Lys Asp Leu Ala Pro Ser Ile Ile Lys Pro Ile25 30 35 aat ggt aca aac tca acc gct tgg gaa cct caa gtt att caa caa aag1036 Asn Gly Thr Asn Ser Thr Ala Trp Glu Pro Gln Val Ile Gln Gln Lys 4045 50 55 atg ccc gaa agt atg aga gtg ccg aaa gca aca aac tcc act tat caa1084 Met Pro Glu Ser Met Arg Val Pro Lys Ala Thr Asn Ser Thr Tyr Gln 6065 70 cct gaa atc att caa caa aat caa caa aaa aca gaa tcg ata gca aaa1132 Pro Glu Ile Ile Gln Gln Asn Gln Gln Lys Thr Glu Ser Ile Ala Lys 7580 85 aaa cag gct cta caa aat ttt gaa att cca aga gat cct aaa act aat1180 Lys Gln Ala Leu Gln Asn Phe Glu Ile Pro Arg Asp Pro Lys Thr Asn 9095 100 gtg cct gtt tat agc aaa att gat aag ggt ttt tac aaa ggt gat act1228 Val Pro Val Tyr Ser Lys Ile Asp Lys Gly Phe Tyr Lys Gly Asp Thr 105110 115 tac aaa gta cgc aaa ggc gat acc atg ttt ctt att gct tat att tca1276 Tyr Lys Val Arg Lys Gly Asp Thr Met Phe Leu Ile Ala Tyr Ile Ser 120125 130 135 ggc atg gat ata aaa gaa ttg gcc aca cta aat aat atg tct gagcca 1324 Gly Met Asp Ile Lys Glu Leu Ala Thr Leu Asn Asn Met Ser Glu Pro140 145 150 tat cat ctg agt att gga caa gta ttg aaa att gca aat aat attccc 1372 Tyr His Leu Ser Ile Gly Gln Val Leu Lys Ile Ala Asn Asn Ile Pro155 160 165 gat agc aat atg ata cca aca cag aca ata aat gaa tca gag gtgaca 1420 Asp Ser Asn Met Ile Pro Thr Gln Thr Ile Asn Glu Ser Glu Val Thr170 175 180 caa aat aca gtc aat gag aca tgg aat gct aat aaa cca aca aatgaa 1468 Gln Asn Thr Val Asn Glu Thr Trp Asn Ala Asn Lys Pro Thr Asn Glu185 190 195 caa atg aaa ccc gtt gct aca cca aca cat tca aca atg cca atcaat 1516 Gln Met Lys Pro Val Ala Thr Pro Thr His Ser Thr Met Pro Ile Asn200 205 210 215 aaa aca cct cca gcc acc tca aat ata gct tgg att tgg ccaaca aat 1564 Lys Thr Pro Pro Ala Thr Ser Asn Ile Ala Trp Ile Trp Pro ThrAsn 220 225 230 gga aaa att att caa gga ttt tcc agt gct gat gga ggc aataaa ggt 1612 Gly Lys Ile Ile Gln Gly Phe Ser Ser Ala Asp Gly Gly Asn LysGly 235 240 245 att gat att agc ggt tct cgt gga caa gct gtt aat gca gcagct gct 1660 Ile Asp Ile Ser Gly Ser Arg Gly Gln Ala Val Asn Ala Ala AlaAla 250 255 260 gga cga gtt gta tat gcc gga gac gct tta cgt gga tat ggtaat tta 1708 Gly Arg Val Val Tyr Ala Gly Asp Ala Leu Arg Gly Tyr Gly AsnLeu 265 270 275 att att att aaa cat aat gac agt tat tta agt gct tat gcacat aat 1756 Ile Ile Ile Lys His Asn Asp Ser Tyr Leu Ser Ala Tyr Ala HisAsn 280 285 290 295 gaa agt ata ctc gtc aaa gat cag caa gaa gtt aaa gcgggt caa caa 1804 Glu Ser Ile Leu Val Lys Asp Gln Gln Glu Val Lys Ala GlyGln Gln 300 305 310 att gct aaa atg gga agt tct gga aca aac aca atc aaactc cat ttt 1852 Ile Ala Lys Met Gly Ser Ser Gly Thr Asn Thr Ile Lys LeuHis Phe 315 320 325 gaa att cgt tat aaa ggt caa tca gta gat cca atg agatat tta cca 1900 Glu Ile Arg Tyr Lys Gly Gln Ser Val Asp Pro Met Arg TyrLeu Pro 330 335 340 aaa aat taatcctaaa aaaatctgca ccttcatcag ttagttgtttagtccaactt 1956 Lys Asn 345 ttggggtgca gatcatttca gttatcagct ttttattaactattttttga aaattgcatt 2016 aggcaaacgt tttcgttccg ataaaaattc ctttataatgtggtcgtttt ttattttttt 2076 gatggatctt ttctatgtta cacatttttc gtggcacgcccgcattatcc aattttcgtt 2136 taaatcagtt attcagtggt ttcagcaaga taatttacccatt 2179 5 345 PRT Haemophilus somnus 5 Met Lys Lys Phe Leu Pro Leu SerIle Ser Ile Thr Val Leu Ala Ala 1 5 10 15 Cys Ser Ser His Thr Pro AlaPro Val Glu Asn Ala Lys Asp Leu Ala 20 25 30 Pro Ser Ile Ile Lys Pro IleAsn Gly Thr Asn Ser Thr Ala Trp Glu 35 40 45 Pro Gln Val Ile Gln Gln LysMet Pro Glu Ser Met Arg Val Pro Lys 50 55 60 Ala Thr Asn Ser Thr Tyr GlnPro Glu Ile Ile Gln Gln Asn Gln Gln 65 70 75 80 Lys Thr Glu Ser Ile AlaLys Lys Gln Ala Leu Gln Asn Phe Glu Ile 85 90 95 Pro Arg Asp Pro Lys ThrAsn Val Pro Val Tyr Ser Lys Ile Asp Lys 100 105 110 Gly Phe Tyr Lys GlyAsp Thr Tyr Lys Val Arg Lys Gly Asp Thr Met 115 120 125 Phe Leu Ile AlaTyr Ile Ser Gly Met Asp Ile Lys Glu Leu Ala Thr 130 135 140 Leu Asn AsnMet Ser Glu Pro Tyr His Leu Ser Ile Gly Gln Val Leu 145 150 155 160 LysIle Ala Asn Asn Ile Pro Asp Ser Asn Met Ile Pro Thr Gln Thr 165 170 175Ile Asn Glu Ser Glu Val Thr Gln Asn Thr Val Asn Glu Thr Trp Asn 180 185190 Ala Asn Lys Pro Thr Asn Glu Gln Met Lys Pro Val Ala Thr Pro Thr 195200 205 His Ser Thr Met Pro Ile Asn Lys Thr Pro Pro Ala Thr Ser Asn Ile210 215 220 Ala Trp Ile Trp Pro Thr Asn Gly Lys Ile Ile Gln Gly Phe SerSer 225 230 235 240 Ala Asp Gly Gly Asn Lys Gly Ile Asp Ile Ser Gly SerArg Gly Gln 245 250 255 Ala Val Asn Ala Ala Ala Ala Gly Arg Val Val TyrAla Gly Asp Ala 260 265 270 Leu Arg Gly Tyr Gly Asn Leu Ile Ile Ile LysHis Asn Asp Ser Tyr 275 280 285 Leu Ser Ala Tyr Ala His Asn Glu Ser IleLeu Val Lys Asp Gln Gln 290 295 300 Glu Val Lys Ala Gly Gln Gln Ile AlaLys Met Gly Ser Ser Gly Thr 305 310 315 320 Asn Thr Ile Lys Leu His PheGlu Ile Arg Tyr Lys Gly Gln Ser Val 325 330 335 Asp Pro Met Arg Tyr LeuPro Lys Asn 340 345

1. An isolated nucleic acid molecule comprising a coding sequence for animmunogenic H. somnus transferrin-binding protein selected from thegroup consisting of (a) an H. somnus transferrin-binding protein 1 and(b) an H. somnus transferrin-binding protein 2, or a fragment of saidnucleic acid molecule comprising at least 15 nucleotides.
 2. The nucleicacid molecule of claim 1 wherein said molecule comprises a nucleotidesequence having at least about 80% identity to the nucleotide sequenceshown at nucleotide positions 708-2693, inclusive, of FIGS. 1A-1C (SEQID NO:1), or a fragment thereof comprising at least about 15nucleotides.
 3. The nucleic acid molecule of claim 2 wherein saidmolecule comprises a nucleotide sequence having at least about 80%identity to the nucleotide sequence shown at nucleotide positions765-2693, inclusive, of FIGS. 1A-1C (SEQ ID NO:1).
 4. The nucleic acidmolecule of claim 1 wherein said molecule comprises a nucleotidesequence having at least about 80% identity to the nucleotide sequenceshown at nucleotide positions 2891-5803, inclusive, of FIGS. 1A-1C (SEQID NO:1), or a fragment thereof comprising at least about 15nucleotides.
 5. The nucleic acid molecule of claim 4 wherein saidmolecule comprises a nucleotide sequence having at least about 80%identity to the nucleotide sequence shown at nucleotide positions2975-5803, 5 inclusive, of FIGS. 1A-1C (SEQ ID NO:1).
 6. A recombinantvector comprising: (a) a nucleic acid molecule according to claim 1; and(b) control elements that are operably linked to said nucleic acidmolecule whereby said coding sequence can be transcribed and translatedin a host cell, and at least one of said control elements isheterologous to said coding sequence.
 7. A recombinant vectorcomprising: (a) a nucleic acid molecule according to claim 2; and (b)control elements that are operably linked to said nucleic acid moleculewhereby said coding sequence can be transcribed and translated in a hostcell, and at least one of said control elements is heterologous to saidcoding sequence.
 8. A recombinant vector comprising: (a) a nucleic acidmolecule according to claim 3; and (b) control elements that areoperably linked to said nucleic acid molecule whereby said codingsequence can be transcribed and translated in a host cell, and at leastone of said control elements is heterologous to said coding sequence. 9.A recombinant vector comprising: (a) a nucleic acid molecule accordingto claim 4; and (b) control elements that are operably linked to saidnucleic acid molecule whereby said coding sequence can be transcribedand translated in a host cell, and at least one of said control elementsis heterologous to said coding sequence.
 10. A recombinant vectorcomprising: (a) a nucleic acid molecule according to claim 5; and (b)control elements that are operably linked to said nucleic acid moleculewhereby said coding sequence can be transcribed and translated in a hostcell, and at least one of said control elements is heterologous to saidcoding sequence.
 11. A host cell transformed with the recombinant vectorof claim
 6. 12. A host cell transformed with the recombinant vector ofclaim
 7. 13. A host cell transformed with the recombinant vector ofclaim
 8. 14. A host cell transformed with the recombinant vector ofclaim
 9. 15. A host cell transformed with the recombinant vector ofclaim
 10. 16. A method of producing a recombinant H. somnustransferrin-binding protein comprising: (a) providing a population ofhost cells according to claim 11; and (b) culturing said population ofcells under conditions whereby the transferrin-binding protein encodedby the coding sequence present in said recombinant vector is expressed.17. A method of producing a recombinant H. somnus transferrin-bindingprotein comprising: (a) providing a population of host cells accordingto claim 12; and (b) culturing said population of cells under conditionswhereby the transferrin-binding protein encoded by the coding sequencepresent in said recombinant vector is expressed.
 18. A method ofproducing a recombinant H. somnus transferrin-binding proteincomprising: (a) providing a population of host cells according to claim13; and (b) culturing said population of cells under conditions wherebythe transferrin-binding protein encoded by the coding sequence presentin said recombinant vector is expressed.
 19. A method of producing arecombinant H. somnus transferrin-binding protein comprising: (a)providing a population of host cells according to claim 14; and (b)culturing said population of cells under conditions whereby thetransferrin-binding protein encoded by the coding sequence present insaid recombinant vector is expressed.
 20. A method of producing arecombinant H. somnus transferrin-binding protein comprising: (a)providing a population of host cells according to claim 15; and (b)culturing said population of cells under conditions whereby thetransferrin-binding protein encoded by the coding sequence present insaid recombinant vector is expressed.
 21. A vaccine compositioncomprising a pharmaceutically acceptable vehicle and an immunogenic H.somnus transferrin-binding protein selected from the group consisting of(a) an H. somnus transferrin-binding protein 1, (b) an H. somnustransferrin-binding protein 2 and (c) an immunogenic fragment of (a) or(b) comprising at least 5 amino acids.
 22. The vaccine composition ofclaim 21 wherein said transferrin-binding protein comprises an aminosequence having at least about 80% identity to the amino acid sequenceshown at amino acid positions 1-971, inclusive, of FIG. 3 (SEQ ID NO:2),or an immunogenic fragment thereof comprising at least about 5 aminoacids.
 23. The vaccine composition of claim 22 wherein saidtransferrin-binding protein comprises an amino acid sequence having atleast about 80% identity to the amino acid sequence shown at amino acidpositions 29-971, inclusive, of FIG. 3 (SEQ ID NO:2).
 24. The vaccinecomposition of claim 21 wherein said transferrin-binding proteincomprises an amino sequence having at least about 80% identity to theamino acid sequence shown at amino acid positions 1-662, inclusive, ofFIG. 4 (SEQ ID NO:3), or an immunogenic fragment thereof comprising atleast about 5 amino acids.
 25. The vaccine composition of claim 24wherein said transferrin-binding protein comprises an amino acidsequence having at least about 80% identity to the amino acid sequenceshown at amino acid positions 20-662, inclusive, of FIG. 4 (SEQ IDNO:3).
 26. The vaccine composition of claim 21 comprising an H. somnustransferrin-binding protein 1 and an H. somnus transferrin-bindingprotein
 2. 27. The vaccine composition of claim 21 further comprising anH. somnus LppB polypeptide.
 28. The vaccine composition of claim 21further comprising an adjuvant.
 29. A method of treating or preventingH. somnus infection in a mammalian subject comprising administering tosaid subject a therapeutically effective amount of a vaccine compositionaccording to claim
 21. 30. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 22. 31. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 23. 32. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 24. 33. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 25. 34. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 26. 35. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 27. 36. A method of treating or preventing H. somnusinfection in a mammalian subject comprising administering to saidsubject a therapeutically effective amount of a vaccine compositionaccording to claim
 28. 37. A method of producing a vaccine compositioncomprising: (a) providing an immunogenic H. somnus transferrin bindingprotein selected from the group consisting of (a) an H. somnustransferrin-binding protein 1, (b) an H. somnus transferrin-bindingprotein 2 and (c) an immunogenic fragment of (a) or (b) comprising atleast 5 amino acids; and (b) combining said transferrin-binding proteinwith a pharmaceutically acceptable vehicle.
 38. A method of detectingHaemophilus somnus antibodies in a biological sample comprising: (a)providing a biological sample; (b) reacting said biological sample withan immunogenic H. somnus transferrin binding protein selected from thegroup consisting of (a) an H. somnus transferrin-binding protein 1, (b)an H. somnus transferrin-binding protein 2 and (c) an immunogenicfragment of (a) or (b) comprising at least 5 amino acids, underconditions which allow H. somnus antibodies, when present in thebiological sample, to bind to said H. somnus transferrin-binding proteinto form an antibody/antigen complex; and (c) detecting the presence orabsence of said complex, thereby detecting the presence or absence of H.somnus antibodies in said sample.
 39. An immunodiagnostic test kit fordetecting Haemophilus somnus infection, said test kit comprising animmunogenic H. somnus transferrin binding protein selected from thegroup consisting of (a) an H. somnus transferrin-binding protein 1, (b)an H. somnus transferrin-binding protein 2 and (c) an immunogenicfragment of (a) or (b) comprising at least 5 amino acids, andinstructions for conducting the immunodiagnostic test.